BE1030901A1 - Suberin Extraction Method - Google Patents

Abstract

A process for extracting suberin from biomass comprising exposing biomass comprising suberin to a solvent at a pressure and a temperature, said temperature being 92% to 107%, preferably 92% to 106 %, preferably from 92% to 105%, preferably from 92% to 104%, preferably from 92% to 103%, preferably from 92% to 102%, preferably from 92% to 101%, preferably from 92% to 100%, preferably 92% to 99%, preferably 92% to 98%, preferably 92% to 97%, preferably 92% to 96%, of the critical temperature in K of the solvent at said pressure.

Description

1 BE2023/5821

Suberin Extraction Method

Technical field of the invention

The present invention relates to the field of biomass recycling, and more particularly to the field of suberin extraction from biomass.

Background of the invention

The design of efficient processes to exploit agricultural, forestry and agri-food residues is essential to mitigate the environmental impact of these sectors and promote an economy based on biomass. Until now, many routes have been developed for the biorefinery of lignocellulosic biomasses. However, the use of subwoody biomasses from plant barks and storage organs, including tuberous root peels and tubers, has remained rare and has mainly focused on the extraction of starch, secondary metabolites and lignin or on energy production.

Around 359 million cubic meters of wood bark corresponding to an average of 190 million tonnes are generated each year and are composed of polysaccharides, lignin and suberin in highly variable concentrations. Potato peels account for between 70 and 140 thousand tonnes while cassava peels represent up to 40 million tonnes of raw material generated each year, partly due to suboptimal peeling. They have a composition similar to wood bark, although it can also vary between species and genotypes.

Current processing routes for potato and cassava peels are animal feed, combustion and residual starch extraction.

Therefore, these by-products are little exploited despite the fact that they could be valuable sources of various polymers such as cutin and suberin. In particular, suberin represents a potential of several million tons of biopolyester enriched with long-chain polyfunctional fatty acids. As fatty acid production capacity is stagnating and demand is expected to continue to grow over the next few years, industrial extraction of suberin from suberowoody biomass

2 BE2023/5821 available in agricultural by-products such as peels would make it possible to diversify the supply of fatty acids and compounds derived from new sources without competing with land use.

Although subwoody biomass is usually found in by-products of the timber industry, storage tubers and roots represent a valuable source of suberin and a unique opportunity to reach new markets by converting their by-products such than the peels in molecules with high added value. However, the exploitation of suberin requires the development of efficient extraction methods and a diversification of the few applications currently available for extracted suberin.

Several methods, including transesterification, hydrogenation, saponification, and ionic liquids, have been described to extract suberin.

Although each method has advantages, none of them meets the three criteria necessary to facilitate industrial implementation: "() a minimum number of extraction steps is required, (ii) the use of products Chemical and/or aggressive conditions should be avoided, and (iii) ideally, well-established operations in the chemical industry should be implemented for upscaling the process. Adding a catalyst increases the cost, complexity and amount of effluent generated during extraction. It is therefore necessary in the art to have new extraction methods that at least partially overcome one or more of the problems. mentioned above.

Summary of the invention

It is an object of the present invention to provide good methods for extracting suberin from biomass. An advantage of the present invention is that the extracted suberin can be used to coat foods.

The above objective is achieved by a method, solution, spray, suberin composition and use according to the present invention.

According to a first aspect, the present invention relates to a process for extracting suberin from biomass comprising the exposure of

3 BE2023/5821 biomass comprising suberin to a solvent at a pressure and at a temperature, said temperature being from 82% to 107%, preferably from 92% to 107%, preferably from 92% to 106%, preferably from 92% to 105%, preferably from 92% to 104%, preferably from 92% to 103%, preferably from 92% to 102%, preferably from 92% to 101%, preferably from 92% to 100 %, preferably from 92% to 99%, preferably from 92% to 98%, preferably from 92% to 97%, preferably from 92% to 96%, of the critical temperature in K of the solvent at said pressure.

In embodiments, the solvent may be one or more alcohols, water, one or more polyols, one or more dimethylcarbonates, one or more hydroxy acids, one or more diacids, or a mixture thereof. In certain embodiments, the extraction of the first aspect makes it possible to obtain the corresponding ester/ether of the extracted molecules (fatty acid, acylglycerols or poly(acylglycerols)).

In embodiments, the solvent may include an alcohol, supercritical carbon dioxide, or a combination of both.

In embodiments, the alcohol may be selected from methanol, ethanol, n-propanol, isopropanol, and mixtures thereof.

In embodiments, the solvent may be methanol and the temperature is 420K to 549K, preferably 473K to 549K, preferably 473K to 544K, preferably 473K to 539K, preferably 473K to 534K, preferably from 473K to 529K, preferably from 473K to 524K, preferably from 473K to 519K, preferably from 473K to 513.4K, preferably from 473K to 508K, preferably from 473K to 503K, preferably from 473K to 498K, preferably from 473K to 493K, and at a pressure of at least 7.0 MPa. This is advantageous because it allows for lower depolymerization of suberin for a similar yield.

In embodiments, the temperature may be 420K or 473K to 513.4K, the pressure being at most 10.0 MPa, preferably at most 90 MPa. This is advantageous because it allows for lower depolymerization of suberin for a similar yield. In these embodiments, the temperature may optionally be 473K to 495K and the pressure is 8.0 to 10.0 MPa, preferably 8.5 to 9.5 MPa.

4 BE2023/5821

This is advantageous because it allows for lower depolymerization of suberin for a similar yield.

In embodiments, the solvent may be methane! at a temperature above 513.4K and at a pressure of at least 8.0 MPa, preferably at least 9.0 MPa, preferably at least 10.0 MPa, preferably at least 11 .0 MPa, preferably at least 12.0 MPa, preferably at least 13.4 MPa.

In embodiments, the biomass may be exposed to methanol at said pressure and temperature for at least 1 second, preferably for at least 5 seconds, preferably for at least 10 seconds, preferably for at least 15 seconds.

In embodiments, the biomass may be exposed to methanol for no more than 60 minutes, preferably no more than 5 minutes, preferably no more than 2 minutes, preferably no more than 45 seconds.

A short treatment time is advantageous because it allows the extraction to be carried out as a continuous treatment, for example in a tubular reactor.

In embodiments, no catalyst is present during exposure of the biomass to methanol. An advantage of embodiments of the present invention is that a catalyst is not required. However, in other embodiments, a catalyst may be present.

In embodiments, the biomass may be plant biomass. For example, it can be plant residues from forestry activity such as tree bark, cork and sawmill residues. It can also be residues from food processing activities such as peels of tubers and tuberous roots (e.g. potato peels and cassava peels). It may also be plant material derived from secondary growing plant species and containing periderm tissue. The biomass may or may not be rich in polyester.

In embodiments, the biomass may be the peel of a tuber or the bark of a tree.

In embodiments, the tuber may be an emmer tuber, preferably a potato or a cassava.

According to a second aspect, the present invention relates to the use of suberin for the coating of an object. In embodiments, this may be done in order to extend its shelf life (by reducing the rate of water loss, gas exchange and/or microbial growth). In other embodiments, this may be done in order to adjust its permeability (for example, if the object is a semi-permeable membrane). In embodiments, suberin may be extracted by a method according to any embodiment of the first aspect. In embodiments, suberin may be in the form of a solution according to the third aspect, in the form of a spray according to the fourth aspect, may be a composition according to the fifth aspect, or may be an extract of suberin according to the sixth aspect.

In embodiments, the object is a plant and/or is a food.

In embodiments, the plant may be a flower, such as a cut flower. In embodiments, the food may be selected from a fruit, vegetable or tuberous root.

In embodiments, the suberin may be present in a solution comprising ethanol, the suberin being present in an amount of 10 to 50 mg/ml.

Any feature of the second aspect may be as correspondingly described in any other aspect of the invention.

In a third aspect, the present invention relates to a solution for coating foods comprising ethanol, in which suberin is present in an amount of 10 to 50 mg/ml. In embodiments, the solution may be applied to an object by spraying, dipping, or brushing.

Any feature of the third aspect may be as correspondingly described in any other aspect of the invention.

In a fourth aspect, the present invention relates to a spray comprising a solution according to any embodiment of the third aspect.

In a fifth aspect, the present invention relates to a suberin composition comprising less than 0.10%, preferably at

6 BE2023/5821 maximum 0.06%o, preferably maximum 0.05%o glycerol on a dry weight basis. Any feature of the fifth aspect may be as correspondingly described in any other aspect of the invention.

In embodiments, the suberin composition may comprise at least 7.1%, preferably 8.0% to 90.0%, preferably 9.0% to 80.0%, preferably 10, 0% to 70.0%, preferably 11.0% to 60.0%, more preferably 12.0% to 55.0% triacyglycerol, monoacylglycerol, or a combination thereof on a basis of dry weight.

In embodiments, the suberin composition may comprise less than 1.0%, preferably less than 0.8%, preferably less than 0.6%, more preferably less than 0.4% acids a , w- dicarboxylic acids on a dry weight basis.

In embodiments, the suberin composition may comprise less than 12.0%, preferably less than 10.0%, preferably less than 8.0%, preferably less than 6.0%, more preferably less of 5.0% w-hydroxy fatty acids on a dry weight basis.

In embodiments, the suberin composition may comprise less than 25.0%, preferably 5.0% to 24.0%, preferably 6.5% to 23.0%, preferably 8%. at 22.0%, more preferably from 85% to 21.0% non-hydroxy fatty acids on a dry weight basis.

For example, the suberin composition may include less than 0.10% glycerol, at least 7.1% but not more than 90.0% triacyglycerol, monoacylglycerol, or a combination thereof, less than 1.0 % a, w-dicarboxylic acids, less than 12.0% w-hydroxy fatty acids, and less than 25.0% but at least 5.0% non-hydroxy fatty acids on a dry weight basis .

In embodiments, the suberin composition may consist of: - 30 to 70% by dry weight of a mixture comprising less than 0.10% glycerol, at least 7.1% but at most 900% triacyglycerol , monoacylglycerol, or a combination thereof, less than 1.0% a, w-dicarboxylic acids, less than 12.0% w-hydroxy fatty acids, and less than 25.0% but at least 5.0% non-hydroxy fatty acids, and

7 BE2023/5821 - 70 to 30% of other components, in which the proportions are given in relation to the total dry weight of the suberin composition.

Any feature of the fifth aspect may be as correspondingly described in any other aspect of the invention.

In a sixth aspect, the present invention may relate to a suberin extract obtainable by any embodiment of the method of the first aspect.

The inventors hypothesized that the extraction of suberin could be carried out in methanol under supercritical conditions (>513.4 K, 8.22 MPa) to facilitate the fracturing of suberolinous biomass. Experiments were carried out and the suitability of the obtained extracts was then evaluated through the extension of the shelf life of fruits, vegetables or other nutrients that are stored before consumption.

An advantage of embodiments of the present invention is that suberin can be extracted from biomass without the need for a catalyst.

An advantage of embodiments of the present invention is that suberin can be extracted from biomass in an economically viable manner.

An advantage of embodiments of the present invention is that suberin can be extracted from biomass in a single step, without the need for a purification step.

An advantage of embodiments of the present invention is that suberin can be extracted from biomass with minimal waste generation.

An advantage of the embodiments of the present invention is that they provide a cheap and easy solution to mitigate food losses, for example in countries where cassava is grown in large quantities as an industrial or staple crop.

Particular and preferred aspects of the invention are described in the accompanying independent and dependent claims. Features of dependent claims can be combined with

8 BE2023/5821 the features of the independent claims and with the features of other dependent claims, where appropriate, and not only as explicitly stated in the claims.

Although there has been constant improvement, change and evolution of devices in this area, current concepts are considered to represent substantial, new and innovative improvements, including departures from previous practice, which allows to provide more efficient, stable and reliable devices of this nature.

The foregoing features, functionalities and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given by way of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

Brief description of the drawings

Figure 1 shows the general principle of supercritical transesterification (TESC) and its effect on the structure of suberin. Figure 2 shows the mass yield of suberin extract obtained from oak bark (Quercus robur), peels of potato tubers (Solanum tuberosum), peels of cassava tuberous roots (Manihot esculenta ) subjected to a supercritical transesterification (TESC) treatment (518K, 8.3MPa) for 60 minutes. Figure 3 shows the effects of

TESC at 518K 8.3MPa 0-60min on the mass yield of suberin extract and the release of glycerol from cassava. Different letters indicate statistically significant differences (P < 0.05, Tukey HSD, n = 3). 0 minutes means that once the temperature has been reached, the reactor has been shut down. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds.

Figure 4 shows, through a principal component analysis, the impact of temperature, pressure and the physical state of the solvent (PSS, L for liquid, CL for compressible liquid, G for gas, SCF for supercritical fluid) on the extraction and depolymerization of suberin from oak bark using methanol. The yield of suberin extract (Extract

9 BE2023/5821 de sub.), free glycerol (Glycerol) and the COOH/Glycerol molar ratio (Ratio) were used as variables. Temperature and pressure are presented as illustrative variables. O minutes means that once the temperature was reached, the reactor was shut down. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds.

Figure 5 shows the residual mass of blueberries over 202 hours with and without coatings of suberin extract obtained by extraction at 493K 0 min 9MPa (0 min means that once the temperature was reached, the reactor was shut down. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds).

Figure 6 shows the residual mass of cherry tomatoes over 202 hours with and without coatings of suberin extract obtained by extraction at 493K 0 min 9MPa (0 min means that once the temperature was reached, the reactor was shut down This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds).

Figure 7 shows the ATR-FTIR spectra of the biomass studied before treatment, after supercritical transesterification at 518K 60min 8.3MPa (Residue) and the corresponding suberin extract (Extract sub.). 2925 cm-1 and 2850 cm-1 are attributed to the aliphatic chain (C-H stretching and C-H stretching,

CH3); 1735 cm-1 is attributed to suberin (C=O stretching); 1603 cm-1 is attributed to lignin (aromatic stretching); 1511 cm-1 is attributed to lignin (aromatic C=C stretching); the range 1200 - 980 cm-1 is attributed to polysaccharides.

Figure 8 shows the effect of TESC at 518K 8.3MPa 60-240 min on the main component of biomass and the mass yield of Manihot esculenta suberin extract expressed as a percentage of the treated mass of dry peels. AIL = acid insoluble lignin and ASL = acid soluble lignin. The absence of letters means that no statistically significant differences were observed within each component (P < 0.05, Tukey

HSD,n = 3).

Figure 9 shows the rate of reduction in mass loss per 24 hours compared to the control with and without suberin extract coatings for the

10 BE2023/5821 blueberries (left) and cherry tomatoes (right). obtained by extraction at 493K

O min 9MPa (0 min means that once the temperature has been reached, the reactor has been shut down. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds.

In the different figures, the same reference signs refer to identical or similar elements.

Description of illustrative embodiments

The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are not restrictive. In the drawings, the size of some elements may be exaggerated and not drawn to scale for illustrative purposes. The dimensions and relative dimensions do not correspond to actual practical reductions of the invention.

Additionally, the terms first, second, third, and the like in the description and claims are used to distinguish between similar elements and not necessarily to describe a sequence, whether temporally, spatially, in terms of classification, or of any other way. It is understood that the terms so used are interchangeable in appropriate circumstances and that the embodiments of the invention described herein are capable of operating in sequences other than those described or illustrated herein.

Additionally, the terms top, bottom, above, below and the like in the description and claims are used for descriptive purposes and not necessarily to describe relative positions. It is understood that the terms so used are interchangeable in appropriate circumstances and that embodiments of the invention described herein are capable of operating in orientations other than those described or illustrated herein.

The terms "above" and "above" are used synonymously and cover situations with and without physical contact. The term “on” means “above and in physical contact with”.

11 BE2023/5821

It should be noted that the term "comprising", used in the claims, should not be interpreted as being limited to the means listed below; it does not exclude other elements or steps. It should therefore be interpreted as specifying the presence of the listed features, integers, steps or components, but does not preclude the presence or addition of other features, integers, steps, components or groups thereof. The term "comprising" in the context of the invention therefore also includes an embodiment in which no other component is present. Thus, the scope of the expression "a device comprising means A and B" should not be interpreted as being limited to devices composed only of components A and B. This means that, with regard to the present invention, the The only relevant components of the device are A and B.

Reference throughout this specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, occurrences of the expressions “in one embodiment” or “in an embodiment” in different places in this specification do not necessarily all refer to the same embodiment, but may do so. Additionally, the features, Particular structures or features may be suitably combined, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Likewise, it should be noted that, in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped into a single embodiment, figure or description for purposes of simplification of the disclosure and to facilitate the understanding of one or more of the different inventive aspects. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than those expressly set forth in each claim. Rather, as the following claims reflect, the inventive aspects reside in less than all the features of a single

12 BE2023/5821 previously disclosed embodiment. Thus, the claims following the detailed description are expressly incorporated into this detailed description, each claim alone constituting a distinct embodiment of this invention.

Additionally, although some embodiments described herein include some features but not other features included in other embodiments, combinations of features of different embodiments are intended to be within the scope of the invention and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments may be used in any combination.

Additionally, some of the embodiments described herein are described as a method or combination of elements of a method that may be implemented by a processor of a computer system or by other means of performing the function . Thus, a processor with the instructions necessary to implement such a method or element of a method forms a means of carrying out the method or element of a method. Additionally, an element described herein of an embodiment of an apparatus is an example of a means of carrying out the function performed by the element for the purposes of carrying out the invention.

In the description provided here, many specific details are outlined. However, it is understood that embodiments of the invention may be implemented without these specific details. In other cases, well-known methods, structures and techniques have not been detailed so as not to obscure the understanding of this description.

The following terms are provided solely to facilitate understanding of the invention.

In the context of the invention, suberin designates a polymer of biological origin mainly present in higher plants. This waxy organic substance is waterproof. It is mainly present in the cell walls of underground tissues as well as in the bark. Suberin contains two domains: that of polyaliphatics and that of

13 BE2023/5821 polyaromatics essentially formed from hydroxycinnamic acid derivatives.

The exact composition of suberin varies depending on the species. A representation of the structure of suberin is as follows:

To vx Polyaramatic Region

CN

87th 9

Gold

TTET

RR To Subérine

LL u = x >, x

BO X Ie X L

L LL L = x L 4% %; m zede

C5 “Tr Ho x £

Polyaromatic region

Examples:

Ethanol, methanol, acetone, and dichloromethane were purchased from Scharlab. The Jeffamine EDR-148 was supplied by TCI. The N,O-

Bis(trimethylsilyl)trifluoroacetamide:Trimethylchlorosilane (BSTFA:TMCS 99:1).

All reagents and solvents were used as received.

The experiments were carried out on oak bark (Quercus robur), potato peels (Solanum tuberosum var Nicola, Belgium) and cassava peels (Manihot esculenta var Valencia, Costa Rica and var Nase,

Rwanda). The potatoes and cassava were peeled manually and subjected to enzymatic treatment as described by Schreiber, L. et al., Planta 220, 520-530 (2005). The oak bark was obtained from a local sawmill.

Both samples were then dried and ground to 1 mm with a

Sprayer 25 (Fritsch). The samples obtained are then designated as raw.

The crude samples were successively extracted with water, ethanol and dichloromethane in a Soxhlet apparatus. The samples obtained are designated as extracts.

14 BE2023/5821

Extractions were carried out in a batch reactor with self-generated pressure. The same extractions can also be carried out on a larger scale in a tubular reactor (for example, a reactor as described in dos Santos, P. R. S. et al, The Journal of

Supercritical Fluids 126, 25-36 (2017).) with the advantages of reducing operational energy cost (i.e. reducing the amount of solvent to be heated) and equipment cost.

For each suberin extraction, 1 g of dried extracted sample was suspended in methanol and heated to the desired temperature in a 75 mL Parr 5000 Series Reactor System (Parr Instrument Co, IL,

USA). Supercritical transesterification (TESC) and quasi-supercritical transesterification (TEQSC) were carried out at 418, 473, 493, 518, 543 or 553K with a sufficient volume of methanol to reach the desired pressure. Once the temperature has been reached, the heating in the reactor has either been stopped (i.e. a "0 minute" treatment which actually lasts 30 seconds because once the heating in the reactor has stopped, the temperature does not drop below the target temperature after approximately 30 seconds), is maintained for the desired duration. Then, the reactor was air-cooled to 333K, the reaction mixture was vacuum filtered through a sintered glass disk (porosity 16 um - 40 um), the retentate was rinsed twice with 20 ML of methanol preheated to 333K. The filtrates were pooled, adjusted to 100 mL and designated as the liquid fraction, while the retentate is designated as the solid residue. The solid residues were used for FTIR analysis, Klason lignin, and polysaccharide analysis.

The liquid fraction was then evaporated with a rotary evaporator (Rotavapor®, Büchi Labortecknik), recovered successively with water, dichloromethane and acetone three times. Water fractions were pooled and used for glycerol measurement, dichloromethane and acetone fractions were pooled and used for analysis

FTIR and measurement of carboxylic acids.

For each suberin extraction, 30 g of dried raw sample (cassava or oak) was suspended in methanol and heated at 493K for 0 minute with 320 g of methanol in a Parr 5500 series reactor.

15 BE2023/5821 600 mL (Parr Instrument Co, Illinois, USA) controlled by a Parr 4848 system.

Once the temperature was reached, the reactor was shut down and water cooled to 333K. This results in contact between the methanol and the biomass at the target temperature which lasts for approximately 30 seconds before starting to drop.

The reaction mixtures were pooled, filtered and the liquid fraction was dried in a rotary evaporator (Rotavapor®, Büchi Labortecknik) at 60°C.

Mass yields of suberin: The mass yield of the suberin extract was calculated by weighing the residue after drying 10 mL of liquid fraction overnight at 100°C. The results are expressed as a percentage of the initial dry weight.

FTIR: Measurements were carried out on a Vertex 70 spectrometer (Bruker, Germany) equipped with a diamond total reflected attenuation (ATR) device. 16 scans were recorded for each sample over the range of 400 to 4000 cm”! with a resolution of 2 cm”.

Glycerol: Glycerol determination was carried out using a commercial kit (K-GCROLGK®, Megazyme, Ireland).

Carboxylic acids: samples were saponified in ethanolic KOH (0.2 M) for 1 h at boiling temperature. The determination was carried out by titration of the remaining KOH with HCl (0.04 M) compared to a control.

The results are expressed in mmol of carboxylic acid functions per 1 g of treated biomass.

Lignins and polysaccharides: structural composition was determined using well-documented techniques including Klason lignin and acid hydrolysis of polysaccharides followed by derivatization (Schmetz, Q. et al.

Industrial Crops and Products 94, 308-317 (2016)).

Partial identification of the composition of the suberin extract: The liquid fractions obtained from oak (518K 60min 7.5MPa and 493K Omin 9MPa) and cassava var Nase (493K Omin 9MPa) were analyzed for compositional analysis. O min means that once the temperature has been reached, the reactor has been stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds. Firstly, 1 ml of sample was evaporated under nitrogen, dissolved in 300 μl of N,O-

Bis(trimethylsilyl)trifluoroacetamide (BSTFA):Trimethylchlorosilane (TMCS)

16 BE2023/5821 99:1 (Sigma Aldrich®, Germany) and 200 uL of dichloromethane and incubated at 60°C for 1 h. The derivatized samples were then injected into a

GC-MS Agilent 5973N (Agilent Technologies, USA). Chromatograms were analyzed using Enhanced Chemstation D.01.02.16 software (Agilent

Technologies, USA) using the Nist17 and Wiley275 spectral libraries.

Results are expressed as a percentage of peak area over total peak area.

Statistical analysis was carried out using R software version 4.1.2 (2021-11-01) with the agricolae package. A linear regression and a test of

Tukey HSD were performed to assess a statistically significant difference. Principal component analysis (PCA) was performed with the RemdR, factomineR and factoextra packages.

The relationship between the FTIR spectra acquired on the liquid oak fractions and the measurement of the glycerol of these fractions (n=24) was studied in order to determine a prediction equation. The calculations were carried out using

A. First, the FTIR spectra were standardized or derived (1st or 2nd order) or a combination of both. Outliers were filtered from the resulting datasets by computing a partial least squares regression with a Leave One Out (PLSr-LOO) sampling method using the predicted residual error sum of squares (PRESS) of the first local minimum number of components as a criterion. Wavenumbers weakly correlated with the glycerol measurement were filtered from the data sets by computing one hundred iterations of the algorithm

Competitive Adaptive Reweighted Sampling (CARS) - PLSr-LOO using the resulting weight as a criterion. The resulting datasets were calculated by PLSr-LOO. The RPD was considered to evaluate which of the resulting models was better.

Example 1: kinetics of supercritical transesterification (TESC) of suberin

Model extractions were carried out as described in Figure 1 on selected sublinear materials (i.e. oak bark, potato peels and cassava peels), using methanol under conditions supercritical (518K, 8.3MPa) for 60 minutes without

17 BE2023/5821 catalyst supplementation. An extract of the three different materials was obtained, ranging from 6.59% to 20.85% of the processed mass (Figure 2). The use of peels generated the highest extraction ratios, almost twice (20.85%) of the amount previously described in Gandini, A. et al., Suberin:

A promising renewable resource for novel macromolecular materials. Prog.

Polym. Sci 31, 878-892 (2006)) (12.1%). On the other hand, the extraction ratio generated from the bark is lower (6.59%) than that previously described in Gandini, A. et al. (39.7%). FTIR analysis showed the presence of a strong band between 1740 and 1700 cm-1 in the raw material (pretreatment) and the extract, while the band is absent in the posttreatment material (residue) (see figure 7). This band is generally attributed to stretching

C=O of the ester bonds and indicates the suberinic character of the extract produced.

FTIR spectra also revealed other typical suberin bands: symmetric stretching of methylene (C-H) and methyl (C-H, CH3) groups at 2850 cm-1; asymmetric stretching of methylene groups (C-H) at 2925 cm-1; stretching of aromatic rings at 1438, 1462 and 1469 cm-1.

To determine the impact of the extraction procedure on the composition of polysaccharides and lignin, extraction kinetics were carried out on cassava peels between 0 and 60 minutes. Here, O minutes means that once the temperature was reached, the reactor was stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds. The kinetic analysis (Figure 3) showed that the maximum extraction level is reached after the first 30 minutes of the process at 518K 8.3 MPa. The extraction appeared to take place in two phases. During the first phase (i.e., between 0 and 20 minutes), approximately 11.5% of the suberin compounds were extracted. The remaining 18.3% was extracted during the second phase (20 to 60 minutes). This result was corroborated by the measurement of glycerol release, which indicated a sequential release of glycerol (0-20 min, 30-50 min and 60 min) (Figure 3); without being bound by theory, it is believed to suggest that there is either a preferential order between primary aliphatic esters and different acylglycerol esters, or that there are suberin domains exhibiting contrasting resistance to transesterification. This last hypothesis could be linked to suberan, a

18 BE2023/5821 crystal structure identified in suberin illustrated by its resistance to saponification.

A second step (between 60 and 240 minutes) confirmed that after 60 minutes, no significant modification of the yield of the suberin extract, the content of polysaccharides and lignins was observed (see figure 8), which implies that polysaccharides and lignins are not extracted. It is also interesting to note that after 60 minutes of treatment, the lignin content measured in the peels of Manihot esculenta and Solanum tuberosum decreased proportionally to the quantity of suberin extract (see table 1).

Table 1: Composition of lignin in the biomass before and after treatment expressed as a percentage of dry matter. Raw = initial composition; TESC 1h: solid residue after supercritical methanolysis at 518K 60 min 8.3MPa; GARLIC = Lignin Insoluble in acid; ASL = Acid Soluble Lignin. Different letters indicate statistically significant differences (P < 0.05, Tukey HSD test, n = 3).

PO |] Ranges

Good

Raw solanum 57.04 + 2.912 5.74 + 0.708 in

Raw Manihot 41.15 + 6.93? 1.95 + 0.582

Ee TL LS

This result suggests that suberin from the barks is measured as lignins in the raw material.

Example 2: Extraction near the suberin critical point

19 BE2023/5821

In this example, the extraction parameters (temperature, pressure, duration) were optimized in order to reduce energy consumption and control the degree of depolymerization of the suberin extract.

Several extractions were carried out on oak bark to study the effect of temperature (418-553K), pressure (1.1-17.5 MPa) and time (0-60 min) on the extract yield and degree of depolymerization.

The depolymerization state of the suberin extract was determined by measuring both the release of glycerol through transesterification and the molar ratio of ester bonds in the extract.

Under subcritical conditions, a simple increase in temperature (Table 2, treatments 2 and 7) or pressure (Table 2, treatments 3 and 4) did not result in a much higher yield, but a decrease in the molar ratio from carboxylic acids to glycerol (i.e. higher depolymerization). Under supercritical conditions, an increase in pressure (Table 2, treatments 11 and 12) resulted in a higher extraction yield and a higher molar ratio of carboxylic acids to glycerol (i.e., more depolymerization). weak). Remarkably, higher temperatures consistently led to lower yields (Table 2, treatments 8 11 and 13).

Longer treatment (Table 2, treatment 3 and 5; 4 and 6; 7 and 9; 8 and 10) consistently resulted in higher yield and depolymerization for the compressible solvent, while for the other solvent states the yield remained the same. The principal component analysis (Figure 4) highlights the fact that the yields and degrees of depolymerization are strongly influenced by the physical state of the solvent. No suberin was extracted for a temperature of 418K (Table 2) under the conditions tested.

Table 2: Effect of temperature (T in K), pressure (P in MPa), time (min) and physical state of the solvent (PSS, L for liquid, CL for compressible liquid, G for gaseous, SCF for supercritical fluid) on the extraction and depolymerization of suberin from oak bark using methanol. Different letters indicate differences

20 BE2023/5821 statistically = significant (P <= 0.05, Tukey HSD, n = 3).

Tromer Fairy | ayceroipss Cove 1 418 [70] 0 À { 00200 0:06: - (2 473 [7080] 0 L 50+O1 | 040.01% | S501+2000 3 493 181-1001 0 CL 72405 |005+001" | 27554584" 4 493 {>145} G CL { 7.6 +0.56 O0b t 0% 1601 + 10035 493 [81-1007 60 CL | 10.0 +0.16} 0.24 4 0.04% 817% 1295 6 493 [>14.5] 60 CL 104 x=03%1 0240027 40 to 3775 7 518 [7080] 0 G 54+01% 0172003 | 1037 + 220% 8 518 181-100) © SCF 91:03 10.13 + 0.026 | 1154 + 4077 3,518 (70-801 60 G { 46 754016 04820025 | 2624 110° 12 543 [134-1451 © ser 11.3 + 08€ | 0.36 + 0.01°° 518 + 848 13 5583 (81100) O SCF 382025 | 55

Interestingly, this experiment confirmed that transesterification, which occurs at a slow rate under conditions near the critical point, is sufficient to efficiently extract suberin material when complete transesterification is not desired. This observation makes it possible to significantly reduce the amount of energy required to heat the methanol and therefore the cost of the process. It can also be assumed that suberin and suberin fragments subjected to a temperature well above their reported melting temperature (between 56 and 103°C) become increasingly soluble in alcohol and facilitate their migration out of the biomass. Suitable alcohols include methanol, ethanol, propanol, isopropanol or a mixture thereof. Alcohol can also be used mixed with other solvents such as water, for example. Supercritical carbon dioxide could also be used, preferably in combination with an alcohol.

21 BE2023/5821

Table 3: main molecules identified in suberin extract. *

Identification carried out using the NIST17 database; **provisional identification. Results are given as percentage of peak area relative to total peak area. O min means approximately 30 seconds.

Éhêne Mare. et bg sg 32

ES 38 268 3x

TracpprermtitAtH 3.1 3L8 | LS;

PRE EE * A © sss

Monoasyighcwol MAG). 3.7 dB | S.Q.

SY-FCTIERA S-aRST > 5 1 12: I

Sy SCENE S-ciôrST z to 1 2 1 Ve

SVME LINKS] The ; | 388 EN nd

U &-dicarhoxyic acid (AUCH 1.3 to 1 E 3

Hlod pres IRAS 125 POP

SONG 183 5 1 F2 1 ENS

DN Ds po rm re SSSR

Ades close to 35 M1 165

Based on these data, their composition was analyzed by GC-MS (Table 3) to check whether mild extraction conditions favor the extraction of larger structures (i.e. acylglycerols instead of 'Fatty acids). We therefore chose two types of extraction with assumed higher (518K 60min 7.5MPa) and lower (493K Omin 9MPa) depolymerization from oak bark. We also analyzed an extraction from cassava peel (493K Omin 9MPa) to check whether, for similar extraction conditions, different raw materials yield similar extracts. O min means that once the temperature has been reached, the reactor has been stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds. Both types

22 BE2023/5821 extraction were in the subcritical state, respectively gaseous and compressible liquid.

Regarding depolymerization, GC-MS analysis revealed that extractions with the highest free glycerol content also had the lowest triacylglycerols (TAG) content (3.1% versus 11.6% of surface area). total peaks (TPA)), which supports the hypothesis of a higher degree of depolymerization. Mild depolymerization conditions of cassava peels were expected to provide a similar profile given their low glycerol release (Table 2). However, instead of TAG, we observed a significant proportion of monoacylglycerols (52% of TPA).

Overall, the profiles show a high amount of fatty acids

C16 to C19 (Table 3), from 10.9% to 25.9% of the total peak area (TPA), in the three selected suberin extracts. This observation suggests the occurrence of hydrolysis rather than alcoholysis (i.e. transesterification) under the conditions used for extraction. Triglyceride hydrolysis has been previously reported as a first step in biodiesel production when subjected to supercritical water conditions. Such hydrolysis was not expected because the aqueous phase during extraction was limited to residual sample water and methanol and considered negligible.

Without being bound by theory, it is thought that the preferential occurrence of hydrolysis in oak (25.9% of TPA) compared to cassava (10.9% of TPA) could be due to a difference in their structure of suberin. The molar ratio of carboxylic acids to glycerol in oak (bark) and cassava (peels) extracts subjected to the same treatments revealed a higher proportion of carboxylic acids to glycerol in oak compared to cassava (Table 4).

Table 4: Effect of temperature (T in K), pressure (P in MPa), time (min) and physical state of the solvent (PSS, CL for compressible liquid, G for gas) on the Extraction and depolymerization of suberin from oak bark (E-Qr) and cassava peels (E-Me) using methanol. Different letters indicate differences statistically

23 BE2023/5821 significant (P < 0.05, Tukey HSD, n = 3).

Biomass - T° - P° - Hard. -PSS | Sub. ext.{%} | Glycerol {mg/g} COOH/GIy.Molar ratio

E-Qr 493 [81-100] O CL | 72x05 | 0.05 +% 001° 2755 + 584°

E-Qr 518 [70-80] 60 G | 46x02 | 10710.03 176 + 13°

E-Me 493 [81100] O CL| 10302 | 02006 281 + 1293

E-Me 518 [70-80] 60 6 | 157249 11003 654 75

This observation is consistent with previous studies that reported higher levels of primary aliphatic esters in cork suberin compared to potato peel suberin (Bento, A. et al.

ACS Sustainable Chemistry and Engineering 9, 15780-15792 (2021)). Similarly, oak bark suberin extract might be composed of a higher proportion of primary aliphatic esters compared to cassava suberin extract. Since these two types of esters are known to have different sensitivity to cleavage, this could explain why we obtained better extraction yields compared to previous studies for peels and worse for bark. Note that we also managed to effectively predict the amount of glycerol released during processing by analyzing the FTIR spectra of the extract (Fig. 6), which can be a useful tool to quickly determine the degree of depolymerization of the extract obtained.

Example 3: use of slightly depolymerized suberin for food coating

Oak and cassava extracts obtained under light depolymerization conditions (493K Omin 9MPa) were selected in order to test industrial applications. O min means that once the temperature has been reached, the reactor has been stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds. The extraction conditions were chosen in order to preserve the native state of suberin and thus preserve its original characteristics. Indeed, previous studies have already demonstrated that slightly depolymerized suberin, obtained using an ionic liquid, is naturally capable of reconstructing a network of

24 BE2023/5821 suberin molecules (Garcia, H. et al, Biomacromolecules 15, 1806-1813 (2014)).

Suberin extracts are generally described in the literature as waxy or oily pastes with a dark brown color, which is consistent with the appearance of suberins obtained under intense depolymerization conditions. In the present invention, the suberin extract obtained under conditions of light depolymerization appeared in the form of glassy brown powder for oak and in the form of brown paste for cassava. This difference indicates a higher glass transition temperature compared to suberin reported in the article by Bento et al.

In addition to their potential compatibility with the suberin extracts obtained in embodiments of the present invention, the experiments on the industrial applications of the suberin extracts obtained in embodiments of the present invention were chosen for their economic, social and environmental importance.

The suberin extracts obtained in embodiments of the present invention can be used as a biological additive in polymers. For example, they can be used in polyurethanes. For example, they can be used in polyurethane foams.

Advantageously, they can be used to increase the viscosity of a formulation for a self-swelling isocyanate-free polyurethane foam.

Advantageously, they can modulate the morphology of polyurethane foams without isocyanate. Advantageously, they can be used to increase the content of biosourced products in a polymer.

However, the inventors of the present invention saw potential for the suberin extracts obtained in embodiments of the present invention in the coating of foods (e.g., fruits and vegetables). In particular, the bio-based nature of the extracts and their waxy properties make them a potential candidate for preventing water loss in foods.

In order to test the potential of suberin extracts to provide a biosourced coating, their application on fruits and vegetables was tested.

The objective was to evaluate the ability of the suberin-based coating to prevent

25 BE2023/5821 water loss and extend the shelf life of blueberries and cherry tomatoes.

To test the suitability of suberin extracts to be used as coatings for fruits and vegetables, blueberries and cherry tomatoes were sprayed with an ethanolic solution of suberin extract obtained under light depolymerization conditions (493K Omin 9MPa) at two different concentrations (i.e. 10 and 50 mg/m)|). (0 min means that once the temperature has been reached, the reactor has been stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds.

The suberin extract of cassava and oak (493K Omin 9MPa) was dried and dissolved in ethanol with concentrations of 10 and 50 mg/mL. 0 min means that once the temperature has been reached, the reactor has been stopped. This results in contact between the methanol and the biomass at the target temperature which lasts approximately 30 seconds. The suspension was sprayed 4 times for 10 seconds on each fruit using a sprayer. A total of 10 cherry tomatoes and 5 groups of 3 blueberries were used for each condition. The simulated treatment consisted of spraying only ethanol and the control did not spray.

The fruits were then stored in an MLR-352H-PA growth chamber (Panasonic, Japan) at 25°C, 50% humidity and one side lit for 202 hours. The fruits were weighed once a day.

The results showed significant differences in water loss from blueberries and cherry tomatoes treated with suberin extracts compared to the mock treatment (Figures 5 and 6). Blueberries showed a 26% and 19% reduction in average daily water loss over 200 hours when coated with an oak suberin extract at 50 mg/ml and a cassava suberin extract at 50 mg/ml, respectively. mg/m! (figure 9) compared to the placebo treatment. The reduction in water loss for cherry tomatoes was 25% after spraying with cassava extracts at 10 mg/ml and 50 mg/ml or oak extract at 10 mg/ml.

The good performance of suberin extracts opens up new opportunities for the supply of biomaterials intended for

26 BE2023/5821 coating applications. Given the importance of post-harvest losses in tropical regions where cassava peels are available in large quantities, cassava suberin extracts could offer cheap and durable coating solutions to extend the shelf life of fruits, vegetables as well as tuberous roots.

It is understood that although preferred embodiments, specific constructions and configurations, and materials have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from of the scope of this invention. For example, any formulas given above are only representative of the procedures that may be used. Functionality can be added or removed from block diagrams and operations can be interchanged between function blocks. Steps may be added or deleted in the methods described in the context of the present invention.

Claims (24)

27 BE2023/5821 Claims 1. A process for extracting suberin from biomass comprising exposing biomass comprising suberin to a solvent at a pressure and a temperature, said temperature being 92% to 107%, preferably 92% to 106%, preferably from 92% to 105%, preferably from 92% to 104%, preferably from 92% to 103%, preferably from 92% to 102%, preferably from 92% to 101%, from preferably from 92% to 100%, preferably from 92% to 99%, preferably from 92% to 98%, preferably from 92% to 97%, preferably from 92% to 96%, of the critical temperature in K of the solvent at said pressure. 2. The process of claim 1, wherein the solvent comprises an alcohol, supercritical carbon dioxide, or a combination of both. 3. The process according to claim 2, in which the alcohol is chosen from methanol, ethanol, n-propanol, isopropanol and mixtures thereof. 4. The process according to any one of the preceding claims, in which the solvent is methanol and in which the temperature is from 473K to 549K, preferably from 473K to 544K, preferably from 473K to 539K, preferably from 473K to 534K, preferably 473K to 529K, preferably 473K to 524K, preferably 473K to 519K, preferably 473K to 513.4K, preferably 473K to 508K, preferably 473K to 503K, preferably 473K at 498K, preferably from 473K to 493K, and at a pressure of at least 7.0 MPa. 5. The method according to claim 4, wherein the temperature is 473K to 513.4K, wherein the pressure is at most 10.0, preferably at most 9.0. 6. The method according to claim 5, wherein the temperature is 473K to 495K and wherein the pressure is 8.0 MPa to 10.0, preferably 28 BE2023/5821 from 8.5 to 9.5 MPa. 7. The process according to any one of claims 1 to 4, in which the solvent is methanol at a temperature above 513.4K and at a pressure of at least 8.0 MPa, preferably at least 9. .0 MPa, preferably at least 10.0 MPa, preferably at least 11.0 MPa, preferably at least 12.0 MPa, preferably at least 13.4 MPa. 8. The process according to any one of the preceding claims, in which the biomass is exposed to methane! at said pressure and at said temperature for at least 1 second, preferably for at least 5 seconds. 9. The process according to claim 8, wherein the biomass is exposed to methanol for at most 60 minutes, preferably for at most 5 minutes, preferably for at most 2 minutes. 10. The process according to any one of the preceding claims, wherein no catalyst is present during the exposure of the biomass to methanol. 11. The method according to any one of the preceding claims, in which the biomass is the bark of a tuber or the bark of a tree. 12. The process according to claim 11, wherein the tuber is a starchy tuber, preferably a potato or a cassava. 13. Use of suberin for coating foods. 14. Use according to claim 13, in which the food is chosen from a fruit, a vegetable or a tuberous root. 29 BE2023/5821 15. Use according to claim 13 or 14, in which the suberin is present in a solution comprising ethanol, in which the suberin is present in an amount of 10 and 50 mg/ml. 16. Use according to any one of claims 13 to 15, in which the suberin is obtained by the process according to any one of claims 1 to 12. 17. A solution for coating foods comprising suberin in a solution comprising ethanol, in which suberin is present in an amount of 10 and 50mg/ml. 18. A spray comprising the solution according to claim 17. 19. A suberin composition comprising less than 0.10%, preferably not more than 0.06%, preferably not more than 0.05%o glycerol on a dry weight basis. 20. The suberin composition according to claim 19 comprising at least 7.1%, preferably 8.0% to 90.0%, preferably 9.0% to 80.0%, preferably 10.0 % to 70.0%, preferably 11.0% to 60.0%, preferably 12.0% to 55.0% triacyglycerol, monoacylglycerol or a combination of both on a dry weight basis. 21. The suberin composition according to claim 19 or claim 20 comprising less than 1.0%, preferably less than 0.8%, preferably less than 0.6%, preferably less than 0.4% of a,w-dicarboxylic acids on a dry weight basis. 22. The suberin composition according to any one of claims 19 to 21 comprising less than 12.0%, preferably less than 10.0%, preferably less than 8.0%, preferably less than 6.0% , of 30 BE2023/5821 preferably less than 5.0% w-hydroxy fatty acids on a dry weight basis. 23. The suberin composition according to any one of claims 19 to 22 comprising less than 25.0%, preferably from 5.0% to 24.0%, preferably from 6.5% to 23.0%, preferably 8% to 22.0%, preferably 8.5% to 21.0%, even more preferably 10.0% to 20.5% non-hydroxy fatty acids on a dry weight basis. 24. A suberin extract obtainable by any of the processes according to claims 1 to 12.