SE543429C2 - Method of preparing plant protein based absorbent material and absorbent material thus produced - Google Patents

Abstract

A method of preparing a plant protein based absorbent material is disclosed, the method comprising the steps of: i. providing a mixture or suspension comprising a liquid and a plant protein, wherein said plant protein is insoluble in the liquid, ii. acylating said plant protein by adding an acylating agent thereto, and iii. obtaining said plant protein based absorbent material, wherein the pH of the mixture or suspension is 10-12, or wherein the pH is adjusted to at least 12 prior to step (ii). A plant based absorbent material obtainable by the method is also disclosed.

Description

lO |\/IETHOD OF PREPARING PLANT PROTEIN BASED ABSORBENT MATERIAL ANDABSORBENT MATERIAL THUS PRODUCED FIELD OF THE TECHNOLOGY The technology proposed herein relates to the field of methods of preparing plantprotein based absorbent materials, thereby providing alternatives to conventional(petroleum based) absorbent materials. More particularly, the technology proposedherein relates to the field of methods of preparing plant protein based absorbentmaterials by acylating plant proteins, and absorbent materials obtained by such methods as well as their use.

BACKGROUND Absorbent materials are used in a wide range of applications where the material'scapacity of absorbing and retaining large amounts of fluids, primarily water, blood orother bodily fluids provide functional benefits. Applications include disposable hygieneproducts, such as diapers and sanitary napkins, and water retention in horticulture, as well as absorbent materials for collecting spills.

While many materials such as tissue paper, cotton and cellulose pulp absorb moderateamounts of water, the absorbing capacity is limited and these materials tend to shed the absorbed water if compressed.

Accordingly, various petroleum based absorbent materials are known, where some arefully petroleum based, such as for example obtained by polymerizing a blend of acrylicacid whereas other materials are partly petroleum based, being formed by grafting acrylonitrile polymers onto a backbone of, for example, starch.

Due to the nature of the products utilizing these absorbent materials, in many casesbeing disposable products to be discarded after having absorbed the respective liquid,significant amounts of petroleum based raw materials are needed and must be disposed of. lO Accordingly, alternatives to the petroleum based raw materials have been sought. Onesuch alternative raw material is protein from biomass, in particular plant protein or fish protein.

US5847089 discloses a protein hydrogel obtained by acylation of lysyl residues in aprotein raw material derived from biomass. ln addition to acylation, the protein rawmaterial is subjected to crosslinking using a dialdehyde. ln one example the acylation of soy protein isolate is performed at a protein concentration of 1-2% in water.

US9643157 discloses a hydrogel composition comprising crosslinked dextran and dextran sulphate. ln order to substitute conventional petroleum based absorbent materials with absorbentbased on protein from biomass, in particular plant based proteins, there arises a needfor method capable of producing such absorbents having a high efficiency, usingsufficiently cheap and easily obtained raw materials, and resulting in absorbent materials having competitive properties as regards absorption and retaining of liquids. lt is accordingly an object of the technology proposed herein to provide a more efficientmethod of producing plant protein based absorbent materials as regards the choice of raw material.

A further object of the technology proposed herein concerns the provision of a moreefficient method of producing plant protein based absorbent materials as regards the steps needed for producing the plant protein based absorbent materials.

Yet a further object of the technology proposed herein concerns the provision of a moreefficient method of producing plant protein based absorbent materials as regards thesteps needed recovering the plant protein based absorbent material produced by the method.

An additional object of the technology proposed herein is to provide a plant proteinbased absorbent material having improved absorbing and retaining capabilities with regards to body fluids. lO SUMMARY At least one of the above mentioned objects are, according to the first aspect of thetechnology proposed herein, achieved by a method of preparing a plant protein basedabsorbent material, comprising the steps of: i. providing a mixture or suspension comprising a liquid and a plant protein, whereinsaid plant protein is insoluble in the liquid, ii. acylating said plant protein by adding an acylating agent thereto, and iii. obtaining said plant protein based absorbent material, .\_,¿_,š.\_\, x ~'\ »ä -'__. __.\_:.~nu ~\ ' \.- w. __ _.. Å _»OO s» .=\.=~=\ v I.. ua.- \-\=.: »vf »<:>\.»\ ._\\= .w -zšfi nu? wšï :så kur: :===.\,A.5š“:~ Accordingly the technology proposed herein is based on the realization that plantprotein can be acylated to yield a plant protein based absorbent material even if theplant protein is not dissolved in a solution, but rather is provided insoluble in a liquid. Asthe plant protein is insoluble in the liquid it becomes easier to handle and separate.Thus the plant protein based absorbent material can be separated or recovered fromthe liquid or from the reaction mixture after the acylation efficiently for example usingcentrifugation, drying or filtration instead of first needing to be precipitated out. Thissaves time and makes the method more efficient. Additionally, any subsequent steps ofcleaning or washing the acylated plant protein are also simplified as the plant protein iseasier to handle and separate for example from a cleaning or rinsing liquid, e.g. using centrifugation.

This result is also surprising in that the acylating agent, Which needs to be able tocontact the plant protein, is evidently capable of reaching and affecting the insoluble plant protein in the liquid.

The method may be used With a wide variety of plant protein including plant proteinsobtained from industrial product streams in which the plant protein is typically obtainedin aggregated form, i.e. where the plant protein is so aggregated that it becomesinsoluble in the liquid. This also provides for obtaining a plant protein based absorbentmaterial while reducing or obviating the need for chemically crosslinking the plant protein when the plant protein based absorbent material is prepared and/or when it is lO used. This increases the efficiency of the method as a crosslinking step may requirearound 12 hours or more to perform, and crosslinking also decreases swelling capacityand can involve toxic Chemicals. Aggregated plant protein can, inter alia, be obtainedfrom industrial product streams, but the aggregation can also be effected deliberately.Aggregated plant protein is accordingly already sufficiently aggregated that mereacylation yields a sufficiently cohesive material capable of absorbing and retainingliquid. The method may however comprise a further step of chemically crosslinking theplant protein for further improving the strength and cohesion of the absorbent materialwhen the absorbent material is used for absorbing liquids in which the plant protein is soluble.

Accordingly, the damage, i.e. aggregation, that is inflicted on a plant protein duringindustrial processing, to obtain a main product to which the protein is an industrial co-product stream, may according to the technology proposed herein, be utilized to makethe process of producing the plant protein based absorbent material more efficient byobviating or reducing the need for chemically crosslinking the plant protein. This notonly simplifies and decreases the cost of preparing a plant protein based absorbentmaterial, it also provides a valuable use for aggregated plant protein obtained fromindustrial process streams, which protein othenNise, due to its content of other non-protein compounds or poor functionality, may not be used in food applications without further treatment. ln the context of the technology proposed herein the term preparing is to be understood as also encompassing the term producing.

Plant protein comprises protein obtained from plants. Plants include, inter alia, tubers (such as potato), cereals and other commercially and non-commercially grown plants.

The plant protein may be obtained from an industrial product stream, i.e. produced orobtained as a product of an industrial process. Examples of plant protein obtained froman industrial product stream include potato protein that is obtained as a product whenpotato tubers are processed for extracting starch, the starch being the main product of the industrial process. lO The plant protein is provided in a mixture or suspension comprising a liquid and theplant protein. The liquid is preferably an aqueous liquid, such as water. For example,when potato protein is obtained as a by-product of starch extraction the potato proteinmay be provided as a suspension corresponding to the potato fruit juice, i.e. the liquid stream resulting after the extraction of the starch.

The liquid should preferably not inactivate the acylating agent or render it ineffective.The plant protein may be rendered insoluble in the liquid by providing a liquid in which the plant protein is not soluble.

The plant protein may alternatively or additionally be rendered insoluble by being sufficiently aggregated so that it is insoluble in the liquid. ln step ii, the acylating agent may be added to the mixture or suspension. Alternatively, the liquid is removed prior to the addition of the acylating agent to the plant protein.

Preferably the plant protein is sufficiently aggregated so as to be insoluble in the liquid.Preferably the plant protein is sufficiently aggregated so that it is insoluble in aqueous liquids, such as water. Water is a suitable liquid for use in the mixture or suspension.

One example of a plant protein suitable for use in the method is potato proteinconcentrate obtained as an industrial product stream from commercial starch extraction. Another example is wheat gluten.

The plant protein being insoluble in the liquid encompasses that the solubility of theplant protein is less than 1 g per liter, more preferably less than 0.1 g per liter of the liquid.

The plant protein may be obtained and provided in sufficiently aggregated form to beinsoluble in the liquid. This is generally the case when the plant protein is obtained froman industrial product stream because industrial processes generally cause damage toplant protein so that it becomes sufficiently aggregated. Examples of such plant proteins are potato protein concentrate and wheat gluten protein. lO Alternatively, the plant protein may be deliberately aggregated, for example by heattreatment including boiling or autoclaving a suspension of the plant protein, optionallywith the addition of acid or base. The technology proposed herein then provides forpreparing absorbent materials also from plant proteins which are not otherwise insoluble in a liquid by the simple step of aggregating them so they become insoluble.

During the acylation step the acylating agent may graft molecules charged carboxylicacid groups onto the plant protein molecules. These carboxylic acid groups, when incontact with a liquid, provide an electric repulsion of different parts of the proteinmolecules from each other, thus leading to swelling of the plant protein based absorbent material.

The acylating agent may be ethylenediaminetetraacetic dianhydride (ED). A preferredacylating agent is succinic anhydride (S), which, as shown in the examples, providesabsorbent material with a high Fluid Swelling Capacity (FSC). Other acylating agentsinclude ethylenediaminetetraacetic acid (E), 1,2,3,4-butanetetracarboxylic acid (B), andcitric acid (C).

The amount of acylating agent used may for example be 20-30 wt. %, such as 25 wt. %relative to the amount of plant protein that is to be acylated. The acylation of the plantprotein is preferably performed at a temperature of at least 20 °C (room temperature)but may be as high as 160 °C.

The plant protein based absorbent material is to be understood as encompassingabsorbent materials comprising plant protein, as well as absorbent materials consistingessentially of plant protein. Preferably the plant protein based absorbent materialcontains at least 70 wt. % plant protein, such as at least 80 wt. % plant protein, or more preferably at least 90 or at least 95, 99 or 100 wt. % plant protein.

The plant protein based absorbent material may advantageously be in the form ofparticles having a particle diameter of 0.01 to 5 mm, such as 0.05 to 1 mm, such as0.05 to 0.5 mm. The desired particle size can be obtained by including a step ofgrinding or othen/vise dividing the plant protein based absorbent material obtained in step (iii) into smaller particles. lO Step iii of the method according to the first aspect of the technology proposed hereinmay further include a step of cleaning the absorbent material obtained after step ii. Theabsorbent material may, for example, be cleaned with water (by dispersing the materialin water and centrifuging or filtering it). lf desired the absorbent material may further beneutralized by adding an acid or base until a neutral pH is obtained, before obtaining the absorbent material in step iii.

The liquid may be an aqueous liquid. Accordingly the method may take as raw materiala plant protein provided directly from an industrial product stream without requiringconcentration, purification or isolation of the plant protein. This is advantageous in thatit further decreases the cost, and increases the available sources of plant protein rawmaterial, for the method. Alternatively, the plant protein can be obtained or provided ina mixture or suspension by mixing dry plant protein with a liquid, preferably an aqueous liquid such as water.

The plant protein should be insoluble in the liquid in the mixture or suspension. Thisallows the plant protein to be easily separated from the liquid after the acylation, for example using filtration or centrifugation as described further below.

Preferably, the plant protein is sufficiently aggregated to be insoluble in the liquid.

The pH of the mixture or suspension is preferably about 11, such as 10 to 12.

The pH may further be adjusted to at least 12 prior to step ii.

Generally, the content of plant protein in the mixture or suspension is from 2 wt. % to90 wt. %, preferably from 2-75 wt. %, more preferably from 2-50 wt. % or 2-40 wt. %,with the remainder being made up preferably by the liquid alone or substantially onlythe liquid ln some embodiments of the method the content of plant protein in the mixture orsuspension is from 2 wt. % to 10 wt. %. Thus the method may use plant protein atlower concentrations thus making it easier to handle the mixture or suspension as it can be pumped or othenNise handled like a liquid. lO ln other embodiments of the method the content of plant protein in said mixture orsuspension is from 10 wt. % to 40 wt. %. Surprisingly, as further evidenced by theexample section, it is possible to acylate the plant protein at a starting plant proteincontent as high as 40 wt. %. This renders the method more efficient as it decreasesrequired capacity for the reaction vessels used for the acylation. lt further decreasesthe amount of liquid that must be removed from the absorbent product to obtain the final absorbent material reducing the energy needed.

Advantageously, in some embodiments of the method, step (iii) of obtaining the plantprotein based absorbent material comprises centrifuging a reaction mixture obtainedfrom step (ii) of acylating said plant protein. This is made possible by using plantprotein that is insoluble in a solvent, e.g. the liquid, in which the acylation may beperformed. Due to the insolubility of the protein there is no need for the conventionaltechniques of precipitating the protein based absorbent material using changes in pH toinduce precipitation; rather the insoluble nature of the plant protein allows the use ofcentrifugation to separate it from the reaction mixture. lt is further contemplated thatother separation methods such as filtration could be used to separate the plant proteinbased absorbent material from the reaction mixture. The process is further simplified inthat, whereas conventional techniques require re-suspending the reaction mixture andadjusting the pH, the centrifugation may be allowed to take place at the pH which wasused during the reaction, i.e. during the acylation. This is especially useful when thecontent of plant protein in the mixture or suspension is from 2 wt. % to 10 wt. %.Therotation speed of the centrifuge may be 2500 to 4500 rpm (corresponding to RelativeCentrifugal Force (RCF) values of about 1100 to 3400). ln alternative embodiments, wherein the content of plant protein in the mixture orsuspension is from 10 wt. % to 40 wt. %, step (iii) of obtaining the plant protein basedabsorbent material may comprise dispersing the acylated plant protein in water afterstep (ii) and allowing it to dry. Thus at these higher concentrations mere drying i.e.allowing the liquid to evaporate, is sufficient to obtain the plant protein based absorbent. lO The plant protein can be obtained using further methods. ln some embodiments step(iii) of obtaining the plant protein based absorbent material comprises lyophilizing theplant protein after step (ii). This provides a plant protein based absorbent materialhaving a porous structure capable of absorbing 9-10 g saline solution (0.9 wt. % saline,i.e. NaCl in water) per gram of the absorbent material after 9 seconds, and having aCentrifuge Retention Capacity (CRC) in said saline solution of 3 g/g at 1230 rpm(corresponding to 270 RCF) for 3 minutes. The absorbing capacity for blood, as testedusing defibronated sheep blood, is comparable to conventional petroleum basedabsorbent materials at 4-8 g/g, and the maximum absorption occurs faster than for the conventional petroleum based absorbent material. ln other embodiments step (iii) of obtaining the plant protein based absorbent materialcomprises drying the plant protein after step (ii) at a minimum of 50°C. This providessolid particles absorbing 20-30 g/g of water and 9-10 g/g of saline solution (0.9 wt. %).The absorption was completed after 100 seconds. The CRC was 3-4 g/g for saline atthe same RCF as above. The absorbing capacity for blood, as tested usingdefibronated sheep blood, is comparable to conventional petroleum based absorbent materials at 4-8 g/g at 30 minutes. ln still further embodiments step (iii) of obtaining the plant protein based absorbentmaterial comprises oven-drying, drum-drying, spray-drying, freeze drying, fluid beddrying, microwave drying, microwave-vacuum drying, vacuum oven drying, shelf dryingor flash-drying said plant protein after step (ii). This provides a particulate solidabsorbent material. The drying may be performed at a temperature of at least 30 °C,such as at least 35, 40 or 50 °C. lf desired the method may further comprise heat denaturing the plant protein at aminimum of 80°C, such as at least 90°C, for at least 30 minutes, prior to step (ii). Theheat denaturing is preferably performed with the plant protein in a mixture orsuspension comprising water and the plant protein, such as an aqueous dispersion.This further increases the cohesiveness of the absorbent material and increasesphysical strength and rigidity. This is because the heat denaturing opens up the protein molecules, thereby rendering them more reactive. lO As stated above the method does not require any step of crosslinking. Thus, inpreferred embodiments the method does not include any step of crosslinking the plantprotein. This saves significant amounts of time, in some cases 12 hours. Further, byavoiding the need for crosslinking there is further no need to use toxic crosslinkingchemicals which are added to the plant protein or that need to be handled or disposed of in the method. ln some embodiments the method however does comprise a step of crosslinking byadding a crosslinking agent to the plant protein. The crosslinking agent may be glutaraldehyde and/or genipin. ln some embodiments the method may further comprise the step of: iv. adding genipin to the plant protein.

This is advantageous in that genipin, which is known as a non-toxic, bio-based,crosslinking agent, surprisingly, and in contrast to the expected effect of reducing theabsorbing capacity of the absorbent material, instead increases the swelling capacity in the present method.

The amount of genipin added may be from 1 to 10 wt. % of genipin, such as 4 wt. % ofgenipin based on the weight of the acylated plant protein. The genipin is preferablyadded after the acylation step (ii), but may alternatively be added before. Genipin ispreferably added at a temperature of at least 35 °C, such as at least 40 °C. The timeneeded for genipin to react with the plant protein may be at least 1 hour, such as 1-4 hour, such as 1-3 hours, such as 2 hours. ln preferred embodiments of the method the plant protein comprises potato protein.Potato protein, especially in the form known as Potato Protein Concentrate (PPC) is areadily available source of non-food grade plant protein and has shown itself to be suitable for the present method.

Other preferred plant proteins comprise wheat gluten protein and soy protein. lO 11 Whereas commercially obtained wheat gluten protein, as is the case with PPC, isaggregated when obtained, soy protein may be provided in aggregated state if needed by aggregating it as described above for being used in the method.

For wheat gluten the content of plant protein in the mixture or suspension is preferablyat least 10 wt. %. However, lower concentrations can be used, preferably if genipin is also added to the plant protein.

Preferably, the industrial product stream is obtained from a starch extraction process,and the industrial product stream is obtained directly after a starch extraction step.Starch is produced in large quantities worldwide. By using plant protein from anindustrial product stream obtained from a starch production process, the industrialproduct streams of the starch extraction can be better utilized. The methodadvantageously allows for using the protein containing industrial product stream directly after starch extraction.

A further aspect of the technology proposed herein concerns a plant protein basedabsorbent material obtained by the method according to the first aspect of the technology proposed herein.

The plant protein based absorbent material may preferably comprise potato protein, i.e.protein derived or obtained from potato. Alternatively, the plant protein basedabsorbent material may comprise wheat gluten protein. The plant protein basedabsorbent material may have a Free Swelling Capacity (FSC) according to any one of the below given values in table 1: Table 1: characteristics of a plant protein based absorbent material Medium FSC (gram media per gram of material) Milli-Q® water (ultrapure water of Type 1 according At least 4 g/g at 300 seconds, more preferably at least 5 g/g at300 seconds, or alternatively at least 8 g/g at 86400 seconds to ISO 3696)0.9 wt. % NaCl solution At least 3 g/g at 10 seconds, or alternatively at least 3,75 g/g at(saline) 600 seconds Defibronated Sheep blood At least 5 g/g of defibronated sheep blood after 30 minutes lO 12 The CRC (Centrifuge Retention Capacity) of the plant protein based absorbent materialfor 0.9 wt. % NaCl solution (saline) may be at least 2 g/g, such as at least 2.5 g/g. ln some cases the plant protein based absorbent material is substantially devoid ofcapillaries, whereas in other cases the plant protein based absorbent material may comprise capillaries.

A further aspect of the technology proposed herein concerns a plant protein basedabsorbent material comprising plant protein, wherein the plant protein is aggregatedand acylated, and wherein the plant protein based absorbent material has a freeswelling capacity (FSC) of at least 3 g of 0.9 wt. % NaCl solution (i.e. 0.9 vvt. % NaCl in water) per gram absorbent material at 10 seconds.

The plant protein based absorbent material may further have a free swelling capacity(FSC) of at least 5 g defibronated sheep blood at 30 minutes, and/or a free swellingcapacity (FSC) of at least 4 g ultrapure water of Type 1 according to ISO 3696 per gram absorbent material at 300 seconds.

The plant protein based absorbent materials according to the technology proposedherein could be used in an absorbent article, as an absorbent material, as anabsorbent material for agricultural water holding and nutrient delivery, or as a carriermaterial for drug delivery. The absorbent material may be used as a degradable waterabsorbent, for example in soil. The absorbent material may further hold and delivernutrients such as nitrogen, phosphorous, trace elements, pesticide and herbicides, etc.Preferably, the absorbent article could be used to absorb blood or urine. As shown inthe example section the plant protein based absorbent material performs at, or close to, conventional petroleum based absorbent materials.

BRIEF DESCRIPTION OF THE FIGURES AND DETAILED DESCRIPTION A more complete understanding of the above mentioned and other features andadvantages of the technology proposed herein will be apparent from the followingdetailed description of preferred embodiments in conjunction with the appended drawings, wherein: lO 13 Fig. 1A shows a first embodiment of the method according to the first aspect ofthe technology proposed herein, Fig. 1B shows a second embodiment of the method according to the first aspectof the technology proposed herein, Fig. 2A shows FSC results obtained in example 1 for different acylating agents, Fig. 2B shows FSC results obtained in example 1 for different acylatingtemperatures, Fig. 2C shows the FSC results obtained in example 1 versus that obtained foruntreated PPC, Fig. 3A shows FSC results obtained in example 2 with and without crosslinking, Fig. 3B shows FSC results obtained in example 2 using different amounts ofacylating agent, Fig. 3C shows FSC results obtained in example 2 depending on the type ofcleaning/washing that is performed after the acylating step, Fig. 4A shows FSC results obtained in example 3 at different proteinconcentrations in water, with/without ED Fig. 4B shows FSC results obtained in example 3 at different proteinconcentrations in 0.9 wt. % saline solution, and Fig. 4C shows FSC results obtained in example 3 using genipin before or after the acylation. ln the below description of the figures the same reference numerals are used todesignate the same features throughout the figures. Further, where present, a “ ' ”added to a reference numeral indicates that the feature is a variant of the feature designated with the corresponding reference numeral not carrying the “ ' “-sign.

Fig. 1A shows a first embodiment of the method according to the first aspect of thetechnology proposed herein. ln this embodiment, termed “wet acylation” the plantprotein is provided as a 2-10wt. % suspension. The suspension is formed in a first step1 by mixing plant protein 2 and water 4. Base, for example NaOH, is added to obtain apH of about 11. Although this embodiment shows the step of forming the suspension 1from dry aggregated plant protein from an industrial product stream, alternatively the aggregated plant protein in the product stream could already be in the form of a lO 14 suspension, or a non-aggregated plant protein could be mixed with water to form asuspension whereafter the suspension was heated, and optionally treated with acid orbase, to cause aggregation of the protein. ln any case the pH of the suspension is preferably adjusted to about 11 using addition of base before acylation.

Following the step of forming the suspension 1 the protein may advantageously beheat denatured at a minimum of 90 °C for at least 30 minutes. After the heat denaturingthe suspension is preferably coo|ed to room temperature, alternatively to 50° C. The pHmay further be increased to 12 by the addition of base 8 in the first stage of theacylation 5. ln the acylation step 5 an acylating agent 10 is added to the suspension.The acylating agent, in this case ED, is preferably added gradually during a timeinterval, such as 1-45 minutes, for example 30 minutes, until 25 wt. % acylating agent,related to the amount of plant protein in the suspension, is reached. Acylation is thenallowed to carry on for 1 to 3 hours, such as 1.5 hours, while further base 8 is added as needed to keep the pH at at least 11, preferably at 12.

Due to the aggregation of the plant protein, the acylated protein is insoluble andadvantageously separated from the suspending water by centrifugation 7 at, forexample, 2500 RPM or about 1100 RCF.

The thus obtained plant protein based absorbent material is then advantageouslycleaned by removal of the supernatant and adding clean water 12. The water 12 shouldpreferably have a pH of about 11 through the addition of a base. Following theresuspension of the absorbent material in the clean water 12 the suspension is oncemore centrifuged to separate the plant protein based absorbent material from thecleaning water. The plant protein based absorbent material is then, after removing thecleaning water and resuspending the material in further water 14, poured on a flatsurface or in a mould. Acid 16 may optionally be added before pouring to neutralize thepH to about 7. Finally the poured plant protein based absorbent material is allowed todry, for example at room temperature or higher, such as for example at 30 °C, 35 °C upto about 50 °C or even 55°C. The drying time obviously varies with the amount of plantprotein based absorbent material that is to be dried and its water content, but maygenerally be in the range of 2-24 h such as 5 hours. The dry plant protein based absorbent material is then advantageously ground 15 into granulate or powder. lO Fig. 1B shows a second embodiment of the method according to the first aspect of thetechnology proposed herein. ln this embodiment, termed “drier acylation” the plant protein is provided as a 40 wt. % suspension forming a mixture or dough.

The mixture is formed in a first step 1' by mixing plant protein 2 and water 4 into adough. Base, for example 1M NaOH, is added to obtain a pH of about 11.

Although the second embodiment shown in Fig. 1B does not utilize a step of heatdenaturing the plant protein, such a step may nevertheless be included in this secondembodiment. lfthe plant protein 2 is not already insoluble in the suspending liquid, forexample by being sufficiently aggregated, it may be heated, and optionally treated with acid or base, to cause it to aggregate.

After forming the mixture a modified acylating step 5' ensues in which the mixture isplaced in a reaction vessel connected to a dean-stark apparatus for condensing anysolvent, in this case condensing water released from the water that was mixed with theplant protein initially and water from the ester formation of the condensation reaction.An acylating agent 10 is added to the suspension. The acylating agent, in this case EDis added until reaching a mass ratio of 0.5:1 (protein:acylating agent). The reactorvessel is heated at a rate of about 1 °C/min from ca. 70° C to 100°C while stirring (ca.30 min) to evaporate water from the mixture. Thereafter the temperature is raised at 10°C/min to the acylation temperature and kept constant for 1 to 3 hours, such as 1.5 hours.

Upon conclusion of the acylation, the mixture, now having the consistency of a paste,was dispersed 17 in water 18 while the pH of the dispersion was raised to neutral bythe addition of base 20, such as NaOH. The dispersed absorbent material was filteredand washed 19 using acetone 22. As an alternative to acetone, ethanol or water at pH11 can be used for the washing step 19. Finally, the plant protein based absorbentmaterial is allowed to dry, for example at about 50 °C. The drying time obviously varieswith the amount of plant protein based absorbent material that is to be dried and itswater content, but may generally be in the range of 2-24 h such as 5 hours. The dryplant protein based absorbent material is then advantageously ground 15 into granulate or powder. 16 Although Figs 1A and 1B show embodiments of the method using PPC as the plantprotein, other plant proteins can be used in the method. One example is wheat gluten(WG). When WG is used as the plant protein, the method is carried out generally asshown in Fig. 1A, however the suspension obtained in the suspension formation step 1should preferably have a content of wheat gluten protein of at least 5 wt. %, preferablyat least 10 wt. %. Further, optionally, the pH of the of the acylated wheat gluten proteinmay be adjusted to 2-3 with HCl (1 M) to flocculate the protein before the centrifugation7. ln this case the cleaning 9 is carried out using water at pH 2-3. Before pouring 11 thepH of the suspension is adjusted (if necessary) to neutral or alkaline (ca. 11) with baseinstead of acid 16, and the suspension is poured on a flat surface and dried as described above.

Further, genipin may be added before or after the acylation step 5.

EXAMPLESExample 1: Acylation of potato protein concentrate from concentrated water suspension 1.1. Background Potato protein concentrate (PPC) is an inexpensive by-product from the agriculturalindustry of starch extraction. Due to the content of non-protein compounds, such asglycoalkaloids, from the industrial process, the protein is not used in food applications.

The protein is in particular aggregated due to the industrial treatment.

The purpose of this example is to investigate the possibility of a fully PPC basedabsorbent material that displays high swelling properties in water, blood and saline solutions.1.2 Materials and methods1.2.1 Materials Commercial potato protein concentrate (PPC) was provided by Lyckeby Starch AB, Sweden, with protein content corresponding to 82 i 2 (Dumas method, Flash 2000 NC lO 17 Analyzer, Thermo Scientific, USA, Nx6.25), and a moisture content of 8.1 i 0.4 %. The PPC powder was used as received.

As for the acylating agents, ethylenediaminetetraacetic dianhydride 98% (ED),ethylenediaminetetraacetic acid 299% (E), succinic anhydride 299% (S), 1,2,3,4-butanetetracarboxylic acid (B) 99%, and citric acid 299.5% (C), were all purchased from Sigma-Aldrich. 1.2.2 Methods The PPC powder was mixed in a beaker with MilliQ quality water (MQW) until ahomogenous protein-rich dough or suspension was formed with a ca. 40 wt. % PPCconcentration. Thereafter, 1M NaOH solution was added dropwise to the dough untilreaching a pH of 11, i.e. for unfolding the PPC. The content from the beaker Was thentransferred to a reaction chamber, connected to a dean-stark apparatus, as well as a mixer. The reactor was placed in an oil bath preheated to 70 °C.

The PPC Was acylated using the five different acylating agents, S, B, ED, C, E, whichwere added through an opening of the reactor. The mass ratio for each acetylationagent was kept constant and 0.5:1 ratio for Protein: acetylation agent. Thereafter, thereactor was covered with an aluminium foil to prevent condensation on the interiorabove the oil bath. The temperature of the reactor was set to increase at a rate of ca. 1°C/min from ca. 70 °C to 100 °C to evaporate the residual water contained in thedough. At the time when a paste-like fluid had formed (after ca. 30 minutes) thetemperature was increased at 10 °C/min from 100 °C to the selected targetedtemperatures of 120, 140 and 160 °C.

As the targeted temperatures were reached, the duration of the reactions was 1.5 h.The approximate time for the evaporation of most of the contained Water in the doughwas ca. 45 min, out of the 1.5 h reaction time (indicated by absence of condensation inthe condensation unit). The reactor was then opened and the Warm paste wastransferred to a beaker containing 200ml i 1ml MQW. The suspension was thoroughlymixed for removal of the unreacted sodium salts of the acylating agent, followed by aneutralization step. The pH of all the suspensions before neutralization (to pH 7) wasca. 2-3. 18 The suspensions were filtered using a filter paper N3 and finally rinsed with acetone(also ethanol and water can be used for rinsing). Due to the increased solubility insome of the treated PPC samples, some suspensions were centrifuged at 1.200 rpm(260 RCF) at the reaction pH (ca. 2-3) before the neutralization and filtration process.These samples are marked with an * in table 2 below. After the centrifugation, thesupernatant was replaced by fresh water, and the mixture was re-dispersed andneutralized. All the clean and neutralized PPC samples were dried overnight at 50 °C.An identically treated PPC sample was produced as reference, i.e. without the additionof any acylating agent (named PPC11). Table 2 summarizes the material protocols tested.

Table 2: materials protocols NAME ACYLATING AGENT TE|\/IPERATURE (°C) PPC11 None 120PPC/S/120 120PPC/S/140 Succininc anhydride 140PPC/S/160* 160PPC/B/120 120 Butanetetracarboxylic acidPPC/B/140* 140 PPC/ED/120 Ethylenediaminetetraacetic 120Dianhydride PPC/C/120 120Citric acidPPC/C/140 140 PPC/E/120 Ethylenediaminetetraacetic 120 acid 1.3. AnalysesWater and saline Free Swelling Capacity (FSC) 19 The free swelling capacity (FSC) of the samples was determined using the “tea-bag”test, according to the standardized procedure of NWSP 240.0.R2.

Three bags filled with 100-200 mg of material per sample being tested. A nonwovenfabric 40x60 mm2, 300-450 mesh (openings of 25-50 um) with heat-sealed edges wasused as the bag, and the filled bags were stored in desiccator for a minimum of 12hprior the test. All bags were hooked to a holding rod and simultaneously immersed in abeaker containing MQW. After the immersion, the bags were placed on a paper towelfor 10 sec for removal of excess of water, and the weight of the bags were recordedafter immersion for 60, 300, 1200, 3600 and 86400 s (Wi). Three empty dry (Wdb) bagswere handled identically to obtain a correction factor (Wu-erik), and then soaked in MQWfor 86400 s (Wwb). The correction factor was obtained as an average of the three replicates. The swelling was calculated according to: Wbiank: Wwb/WdbFSC= ((Wi-(Wb*Wb|,-ank))-(Wd))/Wd Centrifuge retention capacity (CRC) Approximately 100-200 mg of the powder samples were heat-sealed in 40x60 mm2bags of the nonwoven fabric as in FSC. The bags were immersed in 0.9% NaClsolution for 30 min. Thereafter, the bags were centrifuged at 1230 rpm (270 RCF) ontop of glass beads during 3 min and the weight of the bags were recorded (WC). The centrifuge retention capacity of the samples was determined according to: CRC=((WC-(We*Wb|ank)-Wd))/Wd Equally prepared blanks based on empty bags were tested. Three samples were measured and the average is reported.

Blood absorption Blood absorption was determined following the same procedure as for the free swellingcapacity determination. Defibronated sheep blood was used as the test liquid. Theswelling capacity of 100-200 mg of material after 30 min of swelling was determined in duplicates. A commercial SAP was used as a reference material.

Size exclusion liquid chromatography SE-HPLC The protein solubility was evaluated by means of size-exclusion high-performanceliquid chromatography (SE-HPLC) in Waters HPLC equipment, using a BIOSEP SEC-4000 Phenomenex column using a mobile phase of 50:50 water:acetonitrile with 0.1%trifloroacetic acid flowing at 0.2 ml/min. Briefly, 0.5 wt. % sodium dodecyl sulfate (SDS)0.05M NaH2PO4 (pH 6.9) was used as extraction solvent in combination with multipleultra-sonication steps. The first extraction (Ext. 1) was obtained from the supernatant(SN) of a centrifuged dispersion of 16 mg of the ground material in an SDS-phosphatesolution. ln the second extraction (Ext. 2), the centrifuged pellet from Ext. 1 was re-suspended in a new SDS-phosphate solution followed by a 30 s ultra-sonication. Thethird extraction (Ext. 3) of the centrifuged pellet from Ext.2 was performed with freshSDS-phosphate solution and repeated ultra-sonication (30 + 60 + 60 s). Threereplicates were used. The amount of extracted protein was normalized with that of theraw PPC (total extraction from the three extraction steps). The area of the 210nmabsorption chromatogram was arbitrarily divided into polymeric proteins (PP) and monomeric proteins (MP) at 15 minutes of elution. 1.4 Results Fig. 2 shows the water swelling results obtained for the five acylating agents used atthe reaction temperature of 120 °C. lt was observed that a rapid swelling in wateroccurred within the first 60 s of FSC, being the highest for PPC/S/120 and lowest forPPC, with 6 g/g and 2 g/g, respectively (see Fig 2A). At longer times, PPC/S/120 stillshowed the highest FSC reaching a water swelling of ca. 14 g/g (equivalent to ca. 1500% weight increase) after 24 h. S was followed by B, ED, C and E in terms of theswelling uptake. The swelling of the high pH-treated PPC (PPC11) doubled the asreceived PPC. The pH treatment aids protein unfolding and disaggregation as well asgiving some osmotic contribution to the material swelling. This fact is an indication ofthe important role that the protein structure plays when it comes to chemical modification of the protein towards water absorbent properties.

The acylation with succinic anhydride (S) showed an increase in the water swelling byca. 14 % when the reaction temperature was increased to 140 °C (see Fig. 2B). On the contrary, an unexpected material loss was observed for PPC/B/140 (not observed in 21 PPC/B/120) after 30 min of swelling, as seen in Fig. 2B. Further SE-HPLC analysisshowed that this set of conditions resulted in the sample with the highest monomericfraction among the investigated conditions, indicating de-polymerization of the originally aggregated industrial potato protein when treated with B under these conditions.

The FSC in 0.9 wt. % NaCl solution showed that PPC/S/140 reached its maximumswelling already after 10 min of swelling with 4 g/g absorption (in contrast to the 2.5 g/gabsorption for PPC, see Fig. 2C). The decrease in swelling capacity of the material insaline solution is due to the osmotic pressure and charge screening effects that areaffected when mobile ions are present in the liquid. Still, the behaviour and maximumswelling capacity in both water and saline solution of the acylated materials resemblesthe standard swelling ranges for SAPs. Additionally, a CRC of approximately 2 g/g wasobtained for the aforementioned material, double of what is obtained for the referencePPC. The CRC value for PPC/S/140 (see data in Fig. 2C) also indicates that at least50% of the saline solution reported by FSC is held within the bio-based absorbent.

The increase in both saline swelling and CRC compared to the reference sampledemonstrated an increase in the ionic strength and water affinity, respectively, of thebio-based materials herein described. An additional 0.9 wt. % NaCl FSC andcorresponding CRC test made on a commercial SAP revealed that also 50% of thesaline solution is held within the synthetic polymer. Noteworthy is that thefunctionalization process herein applied does not reach the molar amount of carboxylicacid groups present in fossil-based SAPs (e.g. polyacrylic acid) where SAPs rely muchmore on their high content of charges on the polymer to generate high saline swellingvalues (above 40 g/g). Nevertheless, the materials made here were still able to hold thesaline liquid within the acylated PPC (e.g. PPC/S/140) up to superabsorbent ratios.These results show the potential of this chemically modified potato protein industrialproduct stream to be considered as a sustainable and biodegradable absorbentmaterial, utilizing inexpensive acylating agents and readily available and non-foodgrade PPC starting material. The suggested process is also environmentally friendly with potential industrial scalability.

Additional defibronated sheep blood absorption tests showed that PPC/S/120 was ableto swell 5.35 i 0.23 g/g of blood after 30 min, which is approximately half of the blood lO 22 absorption obtained for commercial SAP, this being 10.39 i 3.05 g/g. The defibronatedsheep blood absorption after 30 min for the as received PPC powder was 3.22 i 0.01g/g. These results indicate that these materials have the potential to perform in otherdaily care applications where SAP particulates are used, e.g. sanitary pads and biomedical applications.The SE-HPLC results obtained after the 3 step protein extraction procedure show aclear increase in the MP fraction for the samples PPC/B 140, PPC/C/ 140 and PPC/S/160, see table 3 below: Table 3: SE-HPLC results Sample Total PP % Total MP % Ext1 Ext2 Ext3 % of total % of total % of total extracted for extracted for extracted for PPC PPC PPCPPC 45 55 25 20 55PPC/S/120 63 37 120 120 100PPC/S/140 53 47 220 150 20PPC/S/160* 15 85 220 15 0PPC/B/120 63 37 60 145 130PPC/B/140* 10 90 245 15 0PPC/ED/120 67 33 25 30 100PPC/C/120 65 35 60 125 180PPC/C/140 20 80 245 15 0PPC/E/120 66 34 35 50 185 This indicates that the protein undergoes severe hydrolysis due to the reactionconditions when using B and C at temperatures above 120 °C, and S above 140 °C.These results corresponded to the high material loss observed for the aforementionedsamples and the negative effect in the swelling results, as seen in Fig. 2B.Nonetheless, the balance between monomeric and polymeric fractions for PPC/SA 140was sufficient to improve the swelling properties while keeping the protein networktogether. On the contrary, the increase on the polymeric fraction above 50% for the rest of the samples could be the reason for limited uptake, i.e. the lO 23 aggregation/polymerization of the protein decreases the swelling of the material due torestriction of network expansion. All the samples that showed an increase in the FSC inwater also showed an increase in the total relative amount of extractable proteins(using the unmodified PPC extraction as reference), indicating an increase in the protein solubility.

Without wishing to be bound by theory the increase in the total protein extractionobserved by HPLC for all the treated samples could stem from changes in the proteinmolecular structure, i.e. unfolding, chemical structure changes, molecular weightvariations, etc., which affects the light absorption of the samples. This could influencethe total extraction as the HPLC technique depends on the light absorption propertiesof the proteins. The total protein extracted in the 2nd and 3rd extraction steps (30 s and30 + 60+ 60 s, respectively) was higher for those samples that showed less FSC, i.e.PPC/B 120, PPC/ED120, PPC/E 120, and PPC/C 120.

The SE-HPLC results further show the different aggregation states between thecommercially obtained (aggregated) PPC and the mildly extracted PPC producedthrough ammonium sulphate salting out performed on potato juice. The mildlyextracted potato protein (PPCm) (not aggregated and water soluble) had a SDSextraction (Ext. 1), of 75%, SDS + Sonication 30 s (Ext. 2) of 20% and SDS +Sonication 30+60+60s (Ext. 3) of 5%. ln contrast commercially obtained PPC has Ext.1of 25%, Ext. 2 of 20% and Ext. 3 of 55%. This clearly indicates the high amount ofenergy that has to be put into the system to solubilize the protein fractions of the commercially obtained PPC.

Furthermore, for the PPCm the monomer fraction is 70% of the total extractable proteincontent, whereas for the PPC the monomer fraction is 50% of the total extractable protein, which shows that PPC is aggregated in relation to PPCm.Example 2: Acylation of potato protein concentrate from dilute water suspension2.1 Background The purpose of this example was to investigate the possibility of a potato protein based absorbent material obtained using acylation at lower protein concentrations e.g. 2 wt. lO 24 % ”wet acylation”. A further purpose of this example was to evaluate the effect of adding a crosslinking agent to the material. 2.2 Materials and methods 2.2.1 MaterialsCommercial potato protein concentrate (PPC) was as in example 1ln addition mildly extracted PPC (PPCm) was obtained from potato tubers by extracting the protein with ammonium sulphate precipitation.

Ethylenediaminetetraacetic dianhydride 98% (ED) and Glutaraldehyde (GA, 50% solution) were purchased from Sigma-Aldrich. 2.2.1 Methods PPC was dispersed in a MQw pH 11 solution until a concentration of 2 wt. % proteinwas obtained. While adding the protein to the solution, the pH was continuouslyadjusted to 11 by adding 1M NaOH. Once the protein was homogenously dispersed(ca. 5 min), the dispersion was heated to 90 °C for 30 min to promote denaturation ofthe protein. Thereafter, the beaker was cooled down to room temperature and the pHadjusted to 12. lncremental amounts of ED were added to the beaker corresponding to25 wt. % ED based on the protein content. The reaction was continued for 1.5 h withthe pH maintained at 12 by adding 1M NaOH. The acylated protein was centrifuged at4.500 rpm (3400 RCF) for 5 min, washed with fresh MQw at pH 11 and re-dispersed with a vortexer for 15 min.

At this point a crosslinking agent was optionally added: a) 1 wt. % of glutaraldehyde added dropwise (based on the total amount of protein) The glutaraldehyde treated suspension was left to cure for ca. 12 h at a temperatureabove room temperature (25-45 °C). Both dispersions were poured into petri dishesand dried in a forced air oven overnight, 40° C for the glutaraldehyde treated acylatedPPC, 55° C if no glutaraldehyde was added. The dried films were ground to obtain particles. lO 2.3 Analyses Analyses of the material were performed as described in example 1. 2.4 Results The mildly extracted PPC (PPCm), when acylated, did not yield a material for which aFSC curve could be obtained as the material was lost from the test bags. lt was foundthat PPCm was highly soluble in contrast to the PPC which was aggregated with about40% more strongly bonded ß-sheets compared to PPCm (from FT-IR). Thus the stateof the PPC gives it a sufficiently crosslinked network to be stable on immersion after acylation, and there is no need for further crosslinking.

Fig 3A shows the FSC for saline solution absorption of the acylated PPC with andwithout crosslinking by glutaraldehyde (GA) as well as a reference PPC sample whichis neither acylated nor crosslinked. lt was found that crosslinking impairs the FSC incomparison to acylation only, thus indicating that the commercially obtained, aggregated PPC can be used without chemical crosslinking.

Fig 3B shows the FSC for water for the acylated PPC at different proportions ofacylating agent to PPC. A higher proportion of acylating agent (25 wt. %) provides ahigher FSC.

Fig 3C shows the FSC for saline solution for the acylated PPC (25 wt. % acylatingagent) obtained after different steps of cleaning/washing. The cleaning/washing stepsremove unreacted sodium salts of the acylating agents. This can be performed at roomtemperature. Here “No Clean” refers to no cleaning after the centrifugation, “Clean”refers to resuspension of the absorbent material after centrifugation, and ”“Dya” refersto cleaning by dialysis (3000 Da molecular weight cut off) after pH adjustment to neutral, and “Dya pH 11” refers to dialysis without pH adjustment.

As seen in Fig 3C, a cleaning step is advantageous in increasing the FSC of theabsorbent material, however there is less need to adjust the pH as all “Clean”, “Dyal”and “Dya pH 11” yield similar FSC, a neutral pH however being preferred for daily-careapplication as products which may come into contact with skin should not have highpH. 26 SE-HPLC was performed in order to determine if the water acylation protocol was severely damaging the PPC protein. The results are shown in table 4 below: Table 4: SE-HPLC results Sample Total PP % Total MP % Ext1 Ext2 Ext3 % of total % of total % of total extracted extracted extracted for PPCm for PPCm for PPCmPPCm 27 73 73 23 4PPCm/pH12 38 62 71 13 2PPCm/afterT 56 44 84 5 1Funct/PPCm 48 52 95 2 0Funct/PPCm/GA 68 32 89 6 1PPC 53 46 12 9 23PPC/pH12 51 47 12 9 23PPC/afterT 44 58 17 6 17Funct/PPC 30 65 19 4 10Clean/PPC 53 27 2 4 22Funct/PPC/GA 48 50 0.5 0.4 1SN/after 2 98 39 1 0cleaning The Characterization was carried out on each reaction step the protein is subjected toin achieving the final acylated product. Mildly extracted protein (PPCm) was used asthe reference material. The protein extractability increased through every reaction step,an indication of increased protein solubility. PPCm is a highly soluble protein beforetreatment, contrary to commercially obtained PPC which is highly-aggregated, with ca.40% more strongly bonded ß-sheets than PPCm. The high aggregation state of thecommercial PPC gives a sufficiently crosslinked network for low solubility even beforefunctionalization has taken place. Consequently, there is no need for the addition of crosslinking additives in this system after the acylation of the PPC. Hence, the potential lO 27 of this protein as a candidate in bio-based superabsorbent applications manufactured with less toxic substances is shown.

The HPLC results showed that acylation of commercial PPC led to a decrease inextractability after the acylation. Therefore, an increase in the crosslinked state of theprotein structure, after the acylation, was obtained. This can be proposed as an effectthat influences the functionalization behaviour, promoting an increased crosslink formation and thus less water absorption potential due to reduced network expansion.

Example 3: Acylation of wheat gluten protein from dilute water suspension 3.1 BackgroundThe purpose of this example is to demonstrate the possibility for a wheat gluten proteinbased absorbent material obtained using acylation at concentrations of 10 wt. % through ”wet acylation”. 3.2 Materials and methods 3.2.1 Materials Commercial wheat gluten concentrate (WG) was obtained from Lantmännen ReppeAB, Sweden. The reported protein content is 86,3 i 0,3 (Dumas method, NMKL6:2003, USA, Nx6.25), the moisture content is 6,6 i 0,6 %, fat and ash are 0,9 i 0,1and 0,8 i 0,1 % (2009/152/EU mod and NMKL 173), respectively.

For comparative purposes and to eliminate the influence of the commercial WG on thereactions, mildly extracted gluten was also used (WGm). The extraction of WGm wascarried out by wrapping 20-30 g of wheat flour in a piece of fine cloth and thoroughlywashing it with running water thus removing the starch. The gluten rich fraction wasfrozen at -80 °C and lyophilized for 72 h. The WGm protein content (Dumas method,Thermo Scientific Flash 2000) was 85.5 i 0.6 %.

Ethylenediaminetetraacetic dianhydride 98% (ED) and Glutaraldehyde (GA, 50%solution) was purchased from Sigma-Aldrich. Genipin 98% (G) was purchased from Zhi Xin Biotech company. 28 3.2.1 Methods The acylation of the wheat gluten protein was generally conducted as in example 2,with the main difference that the concentration of the protein was 2 wt. % and 10 wt. %in alternative samples. The pH of the suspension was adjusted to 11. Heat denaturingwas performed at 90 °C. Acylation was performed at pH 12 using the acylating agentED at a final concentration of 25 wt. % relative to the protein content. AftenNards, thepH was lowered to 3.5 to flocculate the protein and remove unreacted ED-salts. Thesuspension was centrifuged at 2500 rpm and the supernatant discarded, the pellet wasresuspended in water and centrifuged, then resuspended and centrifuged once more.The pH was adjusted to neutral or pH 11, and the suspension was poured on a glasspetri-dish, dried overnight in a forced air oven at 50 °C and ground to particles.

Reference samples were similarly treated except for the acylation.

A further experiment was done to study the effect of adding genipin. ln a first experiment 4 wt. % of genipin, according to the amount of WG, was added toa dispersion of 2 wt. % WG at pH 11. The beaker was immediately dipped in a 50 °Cpre-heated water bath while stirring. The suspension gradually changed colourfromyellow to dark blue after ca. 5 min. The WG/G suspension was left to react for 2 h. Afterthis, the suspension was cooled to RT and the acylation proceeded as previouslydescribed. This sample was designated WG/4G/25ED. ln a second experiment, the acylation of WG followed as described above, but beforethe cleaning of unreacted ED salts, the suspension temperature was raised to 50 °Cwhile stirring in a pre-heated water bath, and 4 wt. % (WG mass basis) of G wasadded. The reaction continued for 2 h. The colour of the suspension changed to brownafter 1.5 h of reaction. Thereafter, a cleaning was performed as described above. Thissample was named 2WG/ED/G. 3.3 Analyses Analyses of the material were performed as described in example 1. 3.4 Results lO 29 The functionalization of the WG concentrates by using dilute protein suspensions(2WG/ED, 2 wt. %) led to particles that were not staying within the test bag but leakingout/dissolving in the MQw after the first 10 min, see Fig. 4A. instead, the reference WGparticles 2WG/Ref (which were not acylated) did not show any apparent leakage as themaximum swelling in MQw reached less than 4 g/g and kept stable during the entiretest. Accordingiy, the ED-treatment in the 2wt. % WG protocoi increases the glutenstability in water to a level that does not allow the protein to form a stable network that can expand and hold water without dissoiving to a large extent. in contrast 10WG/ED (10 wt. %) showed a water uptake of ca. 16 g/g and maximum of22 g/g after 30 min and 24 h swelling, respectively. This represents a water swellingimprovement of about 167% - 200% relative to the reference sample at 10 wt. %, and the highest water uptake so far reported for WG particuiate materials.

The 0.9% NaCl swelling, see Fig. 4B, showed that 10WG/ED can reach a maximumuptake of 5 g/g within 10 min. The CRC (g/g) values were WG:1 _52; 2WG/Ref: 1.37;10WG/Ref: 1.41; 10WG/ED: 2.11. The CRC values for the treated WG particles, here10WG/ED were increased 35% compared to WG, with 2.11 g/g or ca. 250%, thusindicating that 50% of the saline solution is held within the treated-WG network.Overall, the MQw, and saline absorption capacity of these samples were low comparedto the absorption capacity measured for commercial SAP used in diapers. Yet, the fastabsorption and MQw/saline uptake obtained allow the material to be considered withinsuperabsorbent poiymer ranges thus having the potential to be used in applicationswhere such SAP poiymers are demanded. The functionalization process did notchange the relative extractable PP (polymeric protein) and PM (monomeric protein)fractions in the 10WG/ED (10wt. %) compared to the as-received WG, beingapproximately 50/50.

The material loss observed in the FSC of the 2WG/ED sample (2 wt. %) was confirmedwith the extractable high molecular weight protein fraction (PP) being ca. 80%. Thishigh PP extractabiiity in 2WG/ED (not observed for 2WG/Ref) is associated with a less aggregated and more soiubie protein network.

To confirm that the Low Concentration (LC) route was not damaging the WG networkwhen using ED, SE-HPLC was performed on dried protein powders extracted aftereach experimental step. Additionally, to make sure that the aggregated state of the as-received commercial WG was not influencing the functionalization process, mildlyextracted WG (WGm) was also studied in the HPLC following the same functionalization route as for 2WG/ED. Results are shown below in table 5.

Table 5: SE-HPLC results (percentages for WG based materials are relative to as received WG and for WGM based materials as produced WGm) Sample Total PP Total MP Ext1 % Ext2 % Ext3 %% % WG 53 47 55 22 23WG/pH12 56 44 81 11 8WG/pH12/90°C 60 40 51 10 39WG/pH12/90°C/ED 65 35 76 19 5WG/pH12/90°C/ED/Clean 78 22 65 28 WGm 55 45 43 27 30WGm/pH12 57 43 70 15 15WGm/pH12/90°C 70 30 48 28 24WGm/pH12/90°C/ED 62 38 79 18 3WGm/pH12/90°C/ED/Clean 70 30 94 5 12WG/Ref 44 56 80 60 302WG/ED 78 22 78 32 81 0WG/ Ref 56 44 20 30 5010WG/ED 52 48 42 16 28 Both the modified WGm and WG samples had approximately the same extractionprofiles. WGm treated under alkali and 90°C conditions gave a higher polymeric proteinextraction (PP) than the WG sample, 70% vs 60% respectively. The functionalizationprocess increases the amount of extracted polymeric fraction (PP) and that thefractions are extracted mainly in the first extraction, indicating an increase in proteinsolubility/extractability, which corresponds to the aim behind the ED treatment, i.e. increasing the proteins affinity to water. lO 31 Overall, the detailed analysis of the reaction steps and the comparison betvveen WGmand WG does not indicate protein depolymerisation/damage to the WG structure due tothe ED treatment. Hence, the increase in solubility/extractability observed for 2WG/EDand the highest total extraction (Ext. 1)for both 2WG/ED and 2WG/Ref shows that theincrease in the protein concentration to at least 10 wt. % is preferred for giving anefficient balance between functionalization and network formation for producing highly swellable WG particles that are cohesive and do not dissolve.

The results for adding genipin, see Fig. 4D, showed that the adding genipin beforeacylation (2WG/G/ED) yielded a maximum of 15 g/g of water swelling, in contrast towhen genipin was added after acylation (2WG/ED/G) which yielded a maximum ofabout 45 g/g of water swelling. lnterestingly, the addition of genipin both prevented thematerial loss which was otherwise present for the 2 wt. % WG samples, and it also increased the swelling capacity.

Claims (17)

1. A method of preparing a plant protein based absorbent material, comprising thesteps of: i. providing a mixture or suspension comprising a liquid and a plant protein, whereinsaid plant protein is insoluble in the liquid, ii. acylating said plant protein by adding an acylating agent thereto, and iii. obtaining said plant protein based absorbent material, 1 .=:.::: :ös ::.-~~ M1., = »w -= _ ' .i :_ _. . _ . .\ V _~~\.-.» “tg (___. \, . \,\¿3»\.~~l:.~\u.=:š.~ u: š::.:š.:,\.~\.~= * ~ OOf:f “I

2. The method according to claim 1, wherein said plant protein is sufficiently aggregated so as to be insoluble in said liquid.

3. The method according to any preceding claim, wherein said liquid is an aqueous liquid.

4. The method according to any preceding claim, wherein the content of plant protein in said mixture or suspension is from 2 wt. % to 10 wt. %.

5. The method according to any of the claims 1-3, wherein the content of said plant protein in said mixture or suspension is from 10 wt. % to 40 wt. %.

6. The method according to any preceding claim, wherein said step (iii) of obtainingsaid plant protein based absorbent material comprises centrifuging a reaction mixture obtained from the step (ii) of acylating said plant protein.

7. The method of claim 5, wherein the content of plant protein in said mixture orsuspension is from 10 wt. % to 40 wt. %, and wherein said step (iii) of obtaining saidplant protein based absorbent material comprises dispersing said acylated plant protein in an aqueous solution, such as water, after step (ii) and allowing it to dry.

8. The method according to any preceding claim, wherein said step (iii) of obtainingsaid plant protein based absorbent material comprises lyophilizing said plant protein after step (ii). lO 33

9. The method according to any of the claims 1-7, wherein said step (iii) of obtaining said plant protein based absorbent material comprises drying said plant protein after step (ii).

10. The method according to claim 9, wherein said step (iii) of obtaining said plantprotein based absorbent material comprises oven-drying, drum-drying, spray-drying,freeze drying, fluid bed drying, microwave drying, microwave-vacuum drying, vacuum oven drying, shelf drying or flash-drying said plant protein after step (ii).

11. The method according to any preceding claim, further comprising heat denaturing the plant protein at at least 80°C prior to step (ii).

12. The method according to any preceding claim, wherein the method does not include any step of crosslinking said plant protein using a crosslinker.

13. The method according any of the claims 1-11, further comprising the step of: iv. adding genipin to the plant protein.

14. The method to any preceding claim, wherein said plant protein comprises potato protein.

15. The method according to any preceding claim, sin. wherein said industrial process stream is obtained from a starch extraction process, and wherein said industrial process stream is obtained directly after a starch extraction step.

16. A plant protein based absorbent material obtained by the method according to any of the preceding claims.

17. A plant protein based absorbent material comprising plant protein, wherein theplant protein is aggregated and acylated, and wherein the plant protein basedabsorbent material has a free swelling capacity (FSC) of at least 3 g of 0.9 wt. % NaCl solution per gram absorbent material at 10 seconds.