EP4646508A1

EP4646508A1 - Plant-based protein coatings

Info

Publication number: EP4646508A1
Application number: EP24743270.1A
Authority: EP
Inventors: Brett Phillip HARDING; Scott Thompson; James Jarratt; Ayaka Kamada; Sabina Silvia Hanel Burmester; James Nelson; Nicholas Mcmullin
Original assignee: Xampla Ltd
Current assignee: Xampla Ltd
Priority date: 2023-06-29
Filing date: 2024-06-28
Publication date: 2025-11-12
Also published as: CN121569075A; AU2024308423A1; WO2025003489A1; WO2025003490A1; KR20260030847A; KR20260030848A; CN121548669A; AU2024310728A1; EP4646509A1

Abstract

The invention relates to a method of coating a substrate, to the coated substrate thus obtained, to a kit for coating a substrate and to the use of the coated substrate for making an item.

Description

PLANT-BASED PROTEIN COATINGS

There is an increasing need to replace polluting plastic packaging with biodegradable alternatives. Paper-based packaging is becoming increasingly popular with consumers because paper or card is highly biodegradable and derived from natural plant sources. Uncoated paper or card packaging is highly biodegradable in many circumstances such as home composting, in fresh water or in soil if disposed of carelessly in the environment. It is also easily recyclable via well-developed commercial recycling systems.

However, paper-based packaging has very poor moisture and oil barrier properties, so its use as packaging material is limited to dry and non-greasy goods. If the product is greasy then this will migrate through the paper permeating to the outside surface of the packaging leaving unsightly marks or damaging the surface it is on. In addition, the goods that are packaged gain little protection from the environment, for example, if the dry product is sensitive to moisture, it will be damaged by ingress of moisture.

Paper-based packaging can be coated with synthetic polymers such as for example polyethylene-based polymers, which, although they can provide good moisture and oil barrier properties, are themselves not biodegradable. If disposed of in the environment the coating will degrade into micro plastics contaminating the soil and water courses. If such coated paper, particularly where thick coatings are used for good barrier properties, enters the paper waste recycling streams it will cause problems such as clogging filters in repulping tanks or reducing the quality of the recycled paper as the polymers are incorporated into it.

Paper-based packaging can also be coated with animal-derived polymers, for example casein and chitosan.

Fibres such as cellulosic fibres or inorganic fibres also benefit from a protective coating to reduce their susceptibility to the environment and attrition. It is again highly desirable that such coatings be biodegradable. Additionally, there is a need for coatings to acts as barriers to protect other substrates from the ingress or loss of moisture, oils and gasses particularly during transport and storage to increase shelf-life. For example, seeds need to be protected but it is highly desirable that the coating be biodegradable so that micro plastic residues do not enter the soil. It is highly desirable to protect fruit and vegetables and it is necessary that the coating is edible and desirable that it is digestible and plant-derived.

Therefore, there remains a need for highly biodegradable, preferably natural plant derived and edible coatings with good water and oil barrier properties. In order for such materials to make a significant impact on the amount of non-biodegradable waste produced, it is necessary for such coatings to be cheap, easy to handle, and to be applied to the material using existing industrial scale coating process. It is also highly desirable that such coatings be very thin to reduce costs and minimise the use of resources and impact on recyclability of materials.

Plant polysaccharides, such as starch, are hydrophilic in nature and their moisture sensitivity means that they do not provide good water barrier properties, particularly at high humidity. In addition, native starch is very prone to retrogradation, both in aqueous solution and when dried into a coating, therefore extensive chemical modification of polysaccharides is needed. Chemically modified starches are typically more water soluble than native starches rendering them unsuitable as water and moisture barriers. Extensive chemical modification will typically result in poor biodegradability, for example cationic starch used as a sizing agent in paper treatment. In addition, native starch aqueous solutions are typically not stable over time making their use in a coating process on an industrial scale more challenging.

Proteins are recognised as an attractive natural polymer since they are readily available, renewable, and biodegradable. Plant-based proteins are particularly attractive polymers as they can be derived from biomass feed stocks to produce bio-based coatings. However, to date their commercial application has been limited by the significant processing challenges associated with their poor solubility in water.

Albumins are the most water-soluble plant-based proteins however they are in low abundance in seed storage material and are lost in the extraction process due to their solubility and are therefore not readily available for industrial processing. Globulins are the most abundant plant-based proteins and their solubility can be tuned by the choice of solvent system to render them suitable for industrial scale processing. However, coatings formed with globulin proteins have poor water barrier properties rendering their use very limited.

Prolamin proteins have been studied as a coating material but they are particularly difficult to incorporate into an aqueous coating process due to their very low solubility. Therefore, more hazardous solvents such as high levels of ethanol and glacial acetic acid are required resulting in environment issues if they are released into the atmosphere or human hazards such as explosion risks. Glutelins are essentially insoluble in water and can only be processed in hazardous solvents.

Attempts have been made to produce plant-based protein coatings from solvent systems where the hazardous solvents are diluted in water, for example WO 2013/010119 uses a water, alcohol and acid solvent system with zein protein, but on an industrial scale hazardous large quantities of solvents will still need to be handled.

Prolamin plant-based proteins are desirable as a coating as they are naturally water- resistant. However, their hydrophobicity means that it is difficult to form a thin consistent homogeneous and even layer on a hydrophilic substrate, such as paper, resulting in a poorly performing coating. Multiple coatings can be applied to improve the integrity of the coating, but such thick coatings increase processing and materials costs.

In addition, the most commonly available plant prolamin, zein, has a vibrant yellow colouring that is imparted to any coated material. This has a negative impact on product aesthetics and the ability to print clearly on such a coating, rendering the final item not consumer acceptable.

It has been surprisingly found that thin water and oil resistant coatings can be created using plant proteins wherein they are applied to a material in a 2-step coating process from 2 different mixtures. The first mixture comprises one or more liquids and one or more plantbased globulin proteins. The second mixture comprises one or more liquids and one or more plant-based prolamin proteins. Preferably, the globulin proteins in the first mixture are dispersed in a mainly aqueous solvent, composed of water and a low level of organic acid, with a low viscosity which spreads very easily on a substrate enabling the formation of a thin homogenous defect-free oil barrier coating. This first coating transforms the irregular and rough substrate surface into a more regular and smother surface. This first coating then facilitates the spreading of the second mixture comprising the one or more plant-based prolamin proteins, thus enabling the formation of a uniform thin second coating and therefore an overall thin coating. This minimises the use of protein, particularly the least water soluble proteins, and hazardous solvents and hence reduces materials and processing costs and manufacturing hazards.

Therefore, in a first aspect the invention relates to a method of coating a substrate comprising the following steps: a. Preparing a first mixture comprising one or more liquids and one or more plantbased globulin proteins; b. Preparing a second mixture comprising one or more liquids and one or more plant-based prolamin proteins; c. Applying the first mixture to at least part of at least a first surface of a substrate to give a first coating; d. Optionally drying the first coating; e. Applying the second mixture on top of at least part of the first coating of the substrate to give a second coating; f. Drying the first coating and/or the second coating.

By “on top of’ it is meant that the first and second coating can be in direct contact with each other. In contrast, it is also possible that there is an intermediate layer between the first and second coatings. Consequently, “on top of” merely defines the order of first and second coatings relative to the substrate without limiting their relationship to each other.

The first mixture and second mixture of the present invention may be prepared by mixing the plant protein into the liquid or vice-versa. By “liquid” it is meant that the respective component is liquid at ambient temperature and ambient pressure.

In preferred aspects of the present invention, solid powdered proteins are fully dispersed into the one or more liquids resulting in a liquid first mixture. In alternative preferred aspects of the present invention, the one or more liquids are fully absorbed by the solid powdered proteins resulting in a solid powder first mixture. The mixing can be conducted at a temperature ranging from 15 °C to 95 °C, more preferably 20°C to 90°C. In exemplified methods the first mixture is held above 70°C for at least 5 minutes, more preferably 80°C for at least 5 minutes. Mixing can be achieved by gentle low shear processes such as stirring, mixing with a paddle stirrer, or processes involving moderate shear such as a rotor stator mixer (e.g.: Silverson mixer) or processes involving high shear for example a high-pressure homogeniser, or sonicator, where mixing is achieved at the microstructure level. Mixing can be achieved using a combination of low, moderate and/or high shear processes. In preferred methods the first mixture is high sheared using a high-pressure homogeniser or sonicator or similar equipment.

In preferred aspects of the present invention, solid powdered proteins are fully dispersed into the one or more liquids resulting in a liquid second mixture. In alternative preferred aspects of the present invention, the one or more liquids are fully absorbed by the solid powdered proteins resulting in a solid powder second mixture. The mixing can be conducted at a temperature ranging from 15 °C to 60 °C, more preferably 20°C to 55°C. In preferred methods the second mixture is held above 40°C for at least 5 minutes, more preferably 50°C for at least 5 minutes. Mixing can be achieved by gentle low shear processes such as stirring, mixing with a paddle stirrer, or processes involving moderate shear such as a rotor stator mixer (e.g. Silveson mixer) or processes involving high shear for example a high- pressure homogeniser or sonicator where mixing is achieved at the microstructure level. Mixing can be achieved using a combination of low, moderate and/or high shear processes. In preferred methods the second mixture undergoes moderate shear using a rotor stator mixer or similar equipment.

In processes with moderate shear, such as a rotor stator mixer (e.g.: a Silverson mixer), the liquid is drawn into the work head by a high-speed rotor where it is intensely mixed in the gap between the rotor and stator, followed by hydraulic shear when the liquid is forced through the stator screen and circulated back into the liquid mixture. This results in a homogeneous material but with limited reduction of particle size.

High pressure homogenisation refers to the process of pumping a stream of liquid through a constriction, e.g. valve, impact surface, narrow pipe or slits causing various degrees of shearing, turbulence and/or cavitation which homogenise the sample, i.e. mix and/or reduce the particle size of any components of the liquid. The high shear forces cause friction between fluid elements and can increase the temperature of the mixture. Optionally high- pressure steam can also be used resulting in additional heating of the liquid. High pressure homogenisation can also pasteurise the liquid prolonging the shelf-life of the mixture obtained. The liquid may be passed through the homogeniser at various levels of pressure and/or kinetic energy once or multiple times to achieve the desired mixture properties. The inlet temperature can be varied but is preferably ambient. The coolant temperature can be varied but it preferably around 5°C. The choice of pressure, inlet and coolant temperature results in a range of liquid temperatures at the nozzle. Preferably the liquid temperature at the nozzle is 20°C to 35°C at 50 MPa, or 35°C to 50°C at 100 MPa or 70°C to 95°C at 250 MPa.

Sonicators or ultraso nicators disrupt particle size through a tip or probe which vibrates very quickly causing bubbles in the mixture that rapidly collapse, i.e.: cavitation. This also generates a significant amount of energy resulting in an increase in temperature of the liquid.

The energy input by moderate or high shear processing, more preferably high shear processing such as high-pressure homogenisation or sonication, breaks up larger aggregates of plant-based proteins within the liquid, resulting in a mixture of soluble protein molecules and insoluble dispersed plant-based protein particles with a smaller particle size than those resulting from low shear processing. The smaller particle size is advantageous in that when the liquid is dried on a substrate to form a coating, there is a more even and consistent spread of the plant-based protein particles. This results in fewer defects, often referred to as pinholes, where the substrate or lower coating layer is exposed and can allow the ingress of moisture or oils, thereby reducing the effectiveness of the coating. The smaller particles size is also advantageous in that it aids the formation of a stable dispersion or solution of the plant-based protein particles in the liquid. Such stable liquids can then be stored for longer periods of time and only require gentle mixing prior to coating a substrate. This is advantageous as the production of the first and second mixtures, to create a kit for coating, is likely to take place in a different manufacturing location to the coating process and with a considerable time lag between them.

Preferably, plant-based proteins dispersed in the first mixture have a particle size with an average diameter, d50, between 0.5 micron and 100 microns, more preferably between 1 micron and 50 microns, even more preferably between 1 micron and 35 microns, most preferably between 2 microns and 25 microns.

Preferably, plant-based proteins dispersed in the second mixture have a particle size with an average diameter, d50, between 0.01 micron and 30 microns, more preferably between 0.1 microns and 25 microns, most preferably between 1 micron and 20 microns. Plant-based proteins are mainly comprised of globular proteins which are storage proteins and can be classified as albumins (soluble in water), globulins (soluble in dilute salt solutions), prolamins (soluble in aqueous ethanol solutions), and glutelins (soluble in dilute acid/alkaline solutions or insoluble in water).

Albumins and globulins are predominately present in all pulses (at greater than 50%) and some pseudo cereals (such as quinoa and amaranth). Globulins represent between about 70 and 78 wt% of the protein found in legume seeds, whereas albumins constitute between about 10 and 20 wt% of the protein. Globulins are the storage proteins of most legume seeds. Globulins have higher molecular weights than albumins and are insoluble in pure water but dissolve in dilute salt solutions. Globulins are typically more water soluble than prolamins.

The storage proteins from different plants can be classified by their sedimentation coefficient in Svedberg units (S). This coefficient indicates the speed of sedimentation of a macromolecule in a centrifugal field. It should be noted however that some small variations of sedimentations are expected depending on the type of plant and/or the extraction protocol employed. Therefore, the sedimentation coefficient is not intended to be restrictive, but rather serve as a useful guide for the classification of the storage proteins.

The major globulins found in pulses are vicilin (7S) and legumin (11 S). The vicilin (7S) has a trimeric structure with molecular mass (MM) of -175-180 kDa and lacks disulfide bridging. In The major globulins found in pulses are vicilin (7S) and legumin (11 S). The vicilin (7S) has a trimeric structure with molecular mass (MM) of -175-180 kDa and lacks disulfide bridging. In contrast, the legumin (11 S) has a hexameric (MM of -340-360 kDa) quaternary structure composed of 6 subunits (MM of ~60kDa) linked by non-covalent interactions. Each subunit pair is comprised of an acidic (MM -40 kDa) and basic (MM -20 kDa) chain joined by a disulfide bond. The ratio of the legumin :vicilin (L/V) is not fixed and may vary among different pulse varieties and species. A third globulin pulse protein is convicilin with 3 or 4 subunits each having a MM of -70 kDa and a sedimentation coefficient of -8S. Convicilin is present in lesser amounts as compared to other globulins. Other globulins include for example 2S globulins, conglutin, sfa, edestin, amandin, concanvalin, cruciferin, helianthinin. The albumins found in pulse proteins are soluble proteins with a variable molecular mass (-12-28 kDa). Albumin proteins include for example 2S albumins, napins, barley trypsin inhibitor and wheat a-amylase inhibitor. In typical commercial protein isolates, there are generally only residual amounts of albumins present as they are generally removed during the protein extraction process.

Globulins are typically obtained from soybean, pea, rice, potato, rapeseed, sunflower, lentil, chickpea, bean, fava bean, mung bean, sunflower seed, pumpkin seed, flax, chia, canola, lupine, alfalfa, moringa, borage, hemp seed, and cotton seed; preferably obtained from pea protein, potato protein, rapeseed protein, and/or sunflower protein.

Prolamins and glutelins make up 85% of protein in the cereal and pseudo cereal families. Prolamins are typically found in wheat, corn, barley and rye whilst glutelins are typically only found in wheat and rice.

Prolamins are high in proline and glutamine amino acid content. They have a relatively high fraction of non-polar functionalities. They are less abundant than globulins and are found across fewer plant species. They include gliadin from wheat, hordein from barley, secalin from rye, zein (alpha, beta, gamma) from corn, kafirin from sorghum, avenin from oats. Prolamins are typically much less water soluble than Globulins.

Preferably, the first mixture comprises one or more plant-based proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, sunflower protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably selected from pea protein, potato protein, rapeseed protein, and/or sunflower protein. Most preferably, the first mixture comprises pea protein.

Preferably, the second mixture comprises one or more proteins selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin. Most preferably, the second mixture comprises zein. In a preferred aspect of the invention, the first mixture comprises the one or more plantbased globulin proteins in an amount of 5 weight % or more.

In another preferred aspect of the invention, the second mixture comprises the one or more plant-based prolamin proteins in an amount of 5 weight % or more.

Plasticisers can be added to either the first or the second mixture to aid processing and to increase coating flexibility. When the substrates that are to be coated are flexible it is important to avoid them cracking when handled, for example when card is folded into boxes, so as not to expose the underlying original uncoated substrate. Plasticisers maybe hydrophobic or hydrophilic. Hydrophilic plasticisers can negatively affect the coatings moisture resistance whilst hydrophobic plasticisers can negatively affect the coatings oil resistance so the level needs to be controlled to balance these features.

Preferably, the one or more hydrophilic plasticisers in the first or second mixture are independently selected from the group consisting of: a) polyols formed by from 1 to 20 repeating hydroxylated units each unit including from 2 to 6 carbon atoms, provided that when the polyol is formed by only one repeating unit it has at least 4 carbon atoms, with the exclusion of sorbitol, b) ethers, thioethers, inorganic and organic esters, acetals and amino-derivatives of polyols formed by from 1 to 20 repeating hydroxylated units each including from 2 to 6 carbon atoms with the exclusion of acetic esters of glycerine, triethyl citrate and tributyl citrate, c) polyol reaction products having from 1 to 20 repeating hydroxylated units each including from 2 to 6 carbon atoms with chain extenders, d) polyol oxidation products having from 1 to 20 repeating hydroxylated units each including from 2 to 6 carbon atoms including at least one aldehydic or carboxylic functional group or mixtures thereof.

A hydrophobic plasticiser can be a water insoluble vegetable oil or wax.

More preferably, the one or more plasticisers in the first or second mixture are independently selected from glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglycerides, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, amino acids, or a mixture thereof, with glycerol and/or oleic acid being most preferred. Preferably, the plasticisers are bio-based and even more preferably plant-derived. Plasticisers can also be added in the form of a mixture with other components, preferably a bio-based and even more preferably a plant-derived mixture.

The level of plasticiser in the first mixture is preferably from 0.5 to 50 wt%, more preferably from 1 to 30 wt%, even more preferably from 1 to 15 wt%, most preferably from 1 to 5 wt%.

The level of plasticiser in the second mixture is preferably from 0.5 to 70 wt%, more preferably from 1 to 50 wt%, even more preferably from 1 to 30 wt%, even more preferably from 1 to 20 wt%, even more preferably from 1 to 15 wt%, most preferably from 1 to 10 wt%.

The ratio of plant-based protein to plasticiser in the first mixture is preferably from 5:1 to 1 :5, more preferably from 3:1 to 1 :3, even more preferably from 3:1 to 1 :1 .

The ratio of plant-based protein to plasticiser in the second mixture is preferably from 6:1 to 1 :6, more preferably from 5:1 to 1 :3, even more preferably from 5:1 to 1 :1 .

In an alternatively preferred aspect of the invention, the first mixture further comprises one or more acids selected from the group consisting of organic and inorganic acids. An acid is a compound which dissociates in water to produce an acidic environment. Inorganic acids usually are considered as strong acids dissociating completely in water, whereas organic acids usually are considered as weak acids dissociating partially in water. Organic acids are preferred for food grade coatings.

In a preferred aspect of the invention, the first mixture further comprises one or more inorganic acids selected from the group consisting of hydrochloric acid, sulphuric acid, sulphurous acid, nitric acid, phosphoric acid. Most preferably, the first mixture comprises hydrochloric acid. Without wishing to be bound by theory it is believed that adjusting the pH of the dispersion with an inorganic acid makes the proteins more soluble and easier to disperse into a uniform slurry as they are further away from the isoelectric point. This enables a more even layer to be formed when the first mixture is applied to a substrate.

In an alternatively preferred aspect of the invention, the first mixture further comprises one or more organic acids selected from the group consisting of acetic acid, an a-hydroxy acid, or a P-hydroxy acid. More preferably, the organic acid is selected from acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, - hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid and carnitine. Even more preferably, the first mixture comprises acetic acid and/or lactic acid, and most preferably lactic acid.

In preferred coatings of the present invention, the organic acid is a volatile organic acid (i.e. those having a boiling point of less than 120 °C at atmospheric pressure), preferably acetic acid. This is because volatile organic acids can be easily removed from a coating mixture during the drying step, such that the final coating contains little, if any, residual organic acid.

In alternatively preferred coatings of the present invention, the organic acid is a low volatility organic acid (i.e. those having a boiling point greater than 120 °C at atmospheric pressure), preferably lactic acid. This is because less volatile organic acids are less easily removed from a coating mixture during the drying step, enabling them to have a dual function as a plasticiser.

The first coatings of the present invention display a useful combination of properties meaning that they are robust and provide oil barrier properties. Increased robustness of the coatings of the present invention can be attributed to the mixing of plant-based protein of the first mixture with an organic acid and the formation of a dense protein first coating upon drying.

Without wishing to be bound by theory, it is believed that when the plant-based protein of the first mixture is added to an aqueous organic acid solution the plant-based proteins partially unfold, resulting in the exposure of hydrophobic amino acids initially buried within the protein native structure. Once partially unfolded, the organic acids are able to interact with the unfolded protein molecules. For example, an organic acid has greater access to protonate amino acid residues, as well as enabling the formation of anion salt bridges that stabilise hydrophobic interactions. In addition, upon heating the first mixture comprising an organic acid at elevated temperatures, protein-protein non-covalent intermolecular contacts are disrupted. Further, it is believed that the application of mechanical agitation to the first mixture comprising an organic acid, for example ultrasonication or high-pressure homogenisation, disrupts large colloidal protein aggregates into smaller ones, as well as disrupting protein intermolecular interactions. Further, it is believed that upon cooling the protein first mixture comprising an organic acid, protein-protein non-covalent intermolecular contacts are enabled, thus promoting the self-assembly of plant-based protein molecules into inter-connected protein aggregates. Drying this plant-based protein first mixture colloidal suspension of protein aggregates on a substrate to form a first coating results in the formation of a dense protein layer with improved robustness and oil barrier properties.

Mixing of the plant-based protein with an organic acid in the first mixture and drying to form a dry coating may result in the plant-based protein having a protein secondary structure with at least 30% intermolecular beta-sheet, at least 40% intermolecular beta-sheets, at least 50% intermolecular beta-sheets, at least 60% intermolecular beta-sheets, at least 70% intermolecular beta-sheets, at least 80% intermolecular beta-sheets, or at least 90% intermolecular beta-sheets.

Mixing of the plant-based protein with organic acid can be done with pure acids, such as glacial acetic acid. However, concentrated acid solutions are dangerous to be handled at large scale. Preferably, the mixing of the plant-based protein with organic acid involves the use of an aqueous organic acid solution. More preferably, the aqueous organic acid solution has a concentration of at least 2% (v/v), preferably at least 3% (v/v), more preferably at least 4% (v/v). Alternatively, the aqueous organic acid solution has a concentration of no more than 50% (v/v), preferably no more than 40% (v/v), more preferably no more than 30% (v/v).

Addition of organic acid to the first mixture of plant-based protein can also result in a more physically stable mixture over time meaning that production at an industrial scale is simpler if larger batches of the first mixture can be made separately and stored until required.

The level of inorganic and/or organic acid in the first mixture is preferably from 1 and 20 wt%, more preferably from 1 .5 and 15 wt%, most preferably from 2 and 10 wt%.

In an alternatively preferred aspect of the invention, the second mixture further comprises one or more organic acids selected from the group consisting of acetic acid, an a-hydroxy acid, or a p-hydroxy acid. More preferably, the organic acid is selected from acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, - hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid, and carnitine.

Addition of organic acid to the second mixture of plant-based protein can also result in a more physically stable mixture over time meaning that production at an industrial scale is simpler if larger batches of the second mixture can be made separately and stored until required.

The level of organic acid in the second mixture is preferably from 0.5 and 50 wt%, more preferably from 1 and 40 wt%, most preferably from 1 to 30 wt%.

In an alternative preferred aspect of the invention the first mixture comprises a plant-based protein that is an alkali-treated plant-based protein, wherein the plant-based protein has been mixed with a base or alkali, so that when the resulting plant-based protein is mixed into water the pH of the dispersion is greater than 7.5, preferably greater than 8, more preferably greater than 9, even more preferably greater than 10. This process generally results in a plant-based protein which is more easily dispersed in water.

In an alternative preferred aspect of the invention the first mixture comprises a plant-based protein that is at a neutral pH, wherein the plant-based protein has been mixed with water, and the pH of the dispersion is between 6.5 and 7.5. This process generally results in a plant-based protein which is less easily dispersed in water.

Water is a preferred liquid in both the first and second mixtures due to its low cost and safe profile.

The one or more liquids in the second mixture are preferably selected from water and alcohols.

The one or more liquids in the first mixture preferably do not contain an alcohol, in particular not ethanol.

In preferred aspects of the present invention, alcohols enhance the solubility and stability of the prolamin plant-based proteins in the second mixture prior to coating. In an alternatively preferred aspect of the invention, the second mixture further comprises one or more alcohols, selected from the group consisting methanol, ethanol, propanol, iso-propanol, butanol, and pentanol. In preferred second mixtures of the present invention, the alcohol is volatile (i.e. those having a boiling point of less than 120 °C at atmospheric pressure), preferably ethanol. This is because volatile alcohols can be easily removed from a coating mixture during the drying step, such that the final coating contains little, if any, residual alcohol.

The first and/or second mixture may additionally comprise waxes. Preferred waxes are made from natural waxes passing the OECD301 B biodegradation screening test, such as bees wax, rapeseed wax, castor wax, candelilla wax, soy wax, palm oil wax or another natural wax, provided that the temperature of exposure does not exceed the wax melting point. In some cases, some paraffin oil-based waxes may also pass OECD301 B.

The first and/or second mixture may additionally comprise titanium dioxide. This can provide both a whitening effect for paper substrates but also can act as a processing aid by reducing the viscosity of the protein mixture.

The first and/or second mixture may additionally comprise a phyllosilicate. Preferably, said phyllosilicate is a serpentine mineral, a clay mineral, a chlorite mineral or a mica mineral, or mixtures thereof. Preferably, said clay mineral is selected from bentonite, kaolinite, pyrophyllite, vermiculite and a smectite (e.g. montmorillonite, cloisite, laponite, hectorite etc.), or mixtures thereof.

Additionally, the phyllosilicates can be used as an intermediary layer between the first and second coatings. Without wishing to be bound by theory it is believed that they behave as a mechanical adhesive between the two coatings and assist with their bonding by through mechanical interlocking.

Additionally, metals or metalloids can be used as an intermediary layer between the first and second coatings, a so called “metallised” layer. The purpose if often to improve oxygen barrier properties of the packaging. Metal atoms, for example Aluminium, or metalloids, for example Aluminium oxides (AIOx), and silicon oxides (SiOx) or an alloy thereof are deposited at the surface. This can also be achieved by transfer-metallisation where an ultrathin layer of metal or metalloid material is deposited onto a support film, which is then placed in contact with the destination substrate, such that the metal or metalloid layer is transferred to said substrate. The first and/or second mixture may additionally comprise biodegradable polymers. Preferably additional biodegradable polymers are selected from starches, algae derived polysaccharides, fungi derived polysaccharides, microbial derived polysaccharides (such as gellan gum and bacterial cellulose), microbial derived polyesters (such as polyhydroxyalkanoates, PHA), chitosan, lignin, cellulose, microfibrillated cellulose (MFC), nanofibrillated cellulose (NFC), microbial nanocellulose or cellulose nanocrystals (CNC). Preferably the additional biodegradable polymers are naturally sourced. Preferably the additional biodegradable polymers are not animal derived.

Starches maybe be native or modified chemically, enzymatically or physically. In preferred mixtures of the present invention, the starch is selected from wheat starch, potato starch, pea starch, waxy potato starch, maize starch, waxy maize starch, high amylose maize starch, tapioca starch, cassava starch, rye starch, sorghum starch, chickpea starch, soy starch, or a mixture thereof, preferably potato starch. In alternative preferred mixtures of the present invention, the starch is a modified starch selected from acid-treated starch, dextrin, alkaline-modified starch, bleached starch, oxidized starch, enzyme-treated starch, maltodextrin, cyclodextrin monostarch phosphate, distarch phosphate, acetylated starch, hydroxypropylated starch, hydroxyethyl starch, starch sodium octenyl succinate, starch aluminium octenyl succinate or cationic starch, or a mixture thereof, preferably acid-treated starch.

Algae derived polysaccharides include red, brown and green algae derived polysaccharide, preferably salts of alginic acid, whether cross-linked or not, carrageenan, furcellaran, agar, ulvans and gums.

Fungi derived polysaccharides includes pullulan.

Cellulose can be extracted from plant or algae sources. Preferably cellulose is in the form of microcellulose, cellulose nanofibres or nanocellulose crystals.

The first and/or second mixture may additionally comprise other auxiliary agents and processing agents, such as, but not limited to, aversive agents such as bitterants (e.g., denatonium salts such as denatonium benzoate, denatonium saccharide, and denatonium chloride; sucrose octaacetate; quinine; flavonoids such as quercetin and naringen; and quassinoids such as quassin and brucine) and pungents (e.g., capsaicin, piperine, allyl isothiocyanate, and resinferatoxin), cross-linking agents, anti-blocking agents, antifoams, antioxidants, bleaching agents (e.g., sodium metabisulfite, sodium bisulfite or others), detackifying agents, extenders, fillers, lubricants, plasticizer compatibilizers, release agents, surfactants, gas-barrier additives (e.g., nanoparticles such as layered silicate-type nanoclays such as sodium montmorillonite), and other functional ingredients, in amounts suitable for their intended purposes.

The amount of such agents can be up to about 50 wt.%, 20 wt.%, 15 wt.%, 10 wt.%, 5 wt.%, 4 wt.% and/or at least 0.01 wt.%, 0.1 wt %, 1 wt %, or 5 wt %, individually or collectively, by weight of the dry coating.

Suitable surfactants may include, but are not limited to, the nonionic, cationic, anionic and zwitterionic classes. Suitable surfactants may include, but are not limited to, polyoxyethylenated polyoxypropylene glycols, alcohol ethoxylates, alkylphenol ethoxylates, tertiary acetylenic glycols and alkanolamides (nonionics), polyoxyethylenated amines, quaternary ammonium salts and quaternized polyoxyethylenated amines (cationics), and amine oxides, N-alkylbetaines and sulfobetaines (zwitterionics). Other suitable surfactants may include, but are not limited to, dioctyl sodium sulfosuccinate, lactylated fatty acid esters of glycerol and propylene glycol, lactylic esters of fatty acids, sodium alkyl sulfates, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, lecithin, acetylated fatty acid esters of glycerol and propylene glycol, and acetylated esters of fatty acids, and combinations thereof. The amount of surfactant in the dry coating may be in a range of from about 0.1 wt.% to about 2.5 wt %, preferably from about 1 .0 wt.% to 2.0 wt.% by weight of the dry coating.

The substrate suitable for coating may be any substrate that requires protection. The substrate material may be a crystalline material, an amorphous material or a fibre-based material. The material may be natural or synthetic; alternatively, it may be plant-based, animal-based or inorganic. Typical crystalline materials include metals, typical amorphous materials include glass, fibre optics and synthetic or bio-based polymers and typical fibrebased materials include paper, textiles, seeds, fruits and vegetables.

In one aspect plant-based protein coatings for fibres are preferred due to the opposite charge between the positively charged plant-based protein and the negatively charged fibre material, for example cellulose, resulting in the formation of a strongly bonded coating. These strong electrostatic interactions, typically result in enhanced properties, including mechanical properties such as strength, stiffness, wear resistance, water resistance, and elasticity. Polycations normally used for electrostatically binding to negatively charged cellulose fibres are animal derived (for example chitosan) or are non-biodegradable (i.e. polyvinylamines).

Fibre-based materials maybe be cellulosic in origin and can be selected from paper (bleached, unbleached, coated in which pores still remain, uncoated, super-calendered), cardboard, wood, fabric or textile, seeds, fruits and vegetables.

Examples of paper materials can include generally thinner, flexible papers, for example useful as wrapping materials, or in making sachets, as well as generally thicker, rigid papers or cardboard (e.g., corrugated cardboard, paperboards, moulded fibreboard), for example useful as boxes, containers, plates, cups, or other storage or food-service items. In this case it is highly desirable that the coating is biodegradable and/or does not affect the recyclability of the paper or cardboard. Test methods commonly used to assess the effect of materials on recyclability include PTS-RH 021 :2012 Cat 1 and Cat 2.

It can also be advantageous that the coating can be heat sealed to enable sachets and boxes to be closed and sealed.

Natural fibres include non-woody fibres, such as cotton, abaca, kenaf, sabai grass, flax, esparto grass, straw, jute, hemp, bagasse, milkweed floss fibres, and pineapple leaf fibres; and woody fibres, such as wood or pulp fibres such as those obtained from deciduous and coniferous trees, including softwood fibres, such as northern and southern softwood kraft fibres, hardwood fibres, such as eucalyptus, maple, birch, and aspen. Pulp fibres may be prepared in high-yield or low-yield forms and may be pulped in any known method, including kraft, sulphite, high-yield pulping methods and other known pulping methods.

The natural fibres to be used in accordance with the present invention may be recycled natural fibres, virgin natural fibres or mixes thereof. Additionally, for good mechanical properties, it may be desirable that the natural fibres be relatively undamaged and largely unrefined or only lightly refined.

Suitable wood materials can be any type of wood commonly used in home, office, and outdoor settings and the material can include furnishings and construction materials. In such cases it is desirable that the coating does not alter negatively the aesthetics and physical properties of the items.

In a particular aspect, the material comprises a porous cellulosic material. A cellulosic material generally includes at least one of cellulose and hemicellulose, and it can further include lignin (e.g., as a lignocellulosic material).

Cellulose-based fibres may include regenerated cellulose fibre such rayon or Cuprammonium rayon, and high pulping yield fibres, unless specified differently. The term “cellulose-based fibres” also includes chemically treated natural fibres, such as mercerized pulps, chemically stiffened or cross-linked fibres, or sulfonated fibres. Also included are mercerized natural fibres, regenerated natural cellulosic fibres, cellulose produced by microbes, the rayon process, cellulose dissolution and coagulation spinning processes, and other cellulosic material or cellulosic derivatives. Other cellulose-based fibres included are paper broke or recycled fibres and high yield pulp fibres including bleached chemo- thermomechanical pulp (BCTMP), chemo-thermomechanical pulp (CTMP), pressure/pressure thermomechanical pulp (PTMP), thermomechanical pulp (TMP), thermomechanical chemical pulp (TMCP), high yield sulphite pulps, and high yield Kraft pulps, all of which leave the resulting fibres with high levels of lignin but are still considered to be natural fibres. High yield fibres are well known for their stiffness in both dry and wet states relative to typical chemically pulped fibres.

Suitable fabric or textile substrates can include any cellulosic materials commonly used in garments or upholstery or otherwise, such as cotton, jute, flax, sisal, hemp, etc.

Suitable seed substrates include those which can be used in domestic, horticultural, or agricultural settings. In this case it is highly desirable that the coating is biodegradable so as not to pollute the environment.

Fruit and vegetable substrates may be fresh or dried or frozen and will benefit from a coating to provide protection during storage and transports to increase their shelf life and reduce spoilage. Fruit and vegetables also benefit from the coating providing a gas barrier to reduce the ripening effects of ethanol produced by other produce stored in close proximity. In this case it is necessary that the coating is edible and highly desirable that it is digestible. Inorganic fibres are often fragile and require protection from the environment. Common examples of such fibres are silica-based fibres such as fibre optics which is very pure and has a very low index of refraction, however they are fragile. Alternatively, plastic fibre optic cables are made from acrylate and polyimides, can be used but these eventually breakup in the environment into harmful micro plastic. Glass fibres also include those used in Fibreglass or mineral wool and Rockwool.

In a preferred aspect of the invention, the substrate material is a fibre-based material. In another preferred aspect of the invention, the substrate material is a cellulosic material.

More preferably, the substrate material is selected from the group consisting of wood, wood pulp, cotton fibres, hemp fibres, jute fibres, sisal fibres, flax fibres, cellulose-based fibres, silica-based fibres, fruits, vegetables, and seeds.

More preferably, the substrate material is selected from the group consisting of paper, cardboard, corrugated board.

A variety of coating methods can be employed depending on the substrate to be coated.

For physically fragile and/or heat-sensitive substrates, such as fruits and vegetables, coating is typically achieved by dip coating or spray coating, with drying at temperatures between 4°C and 50°C, preferably between ambient and 30°C, more preferably at ambient. For drying at lower temperatures this may need to take place over several hours.

For seeds the coating is typically applied in a drum coater, spray coater, rotary coater, fluid bed or extruder. Drying typically is carried out at ambient temperature.

In a preferred aspect of the invention, applying the first and/or second mixture to the substrate is achieved by roller coating, dip coating, slot dies, air knives, or spray coating.

For materials that are not sensitive to heat, pressure or shear, for example paper or fabrics, coating can be undertaken using a variety of equipment including reverse rollers, direct rollers, gravure rollers (direct and reverse), blade over rollers, flexographic equipment, lithographic equipment, slot dies, air knives, spray coating or dip coating. Once the coating has been applied, the wet coated sample is exposed to temperatures between 50°C and 200°C, preferably 70°C and 150°C, more preferably 80°C and 130°C in order to dry it including equipment such as steam cylinders, Yankee Dryers, Flakt Dryers, Infra-red dryers, non-contact dryers (fans) or air flotation dryers.

In a further preferred aspect of the invention, drying of the first coating and/or the second coating is achieved by exposure to air at temperature between 50°C and 250°C for between 0.1 seconds and 10 minutes, more preferably 60°C and 200°C for between 2 seconds and 5 minutes, most preferably between 80 °C and 150°C for between of 6 seconds and 3 minutes. Preferably, drying of the first coating and/or the second coating is achieved by noncontact drying, preferably by using a fan.

When the substrate is paper or cardboard, drying of the first coating and/or the second coating is preferably achieved by exposure to elevated temperatures for a period of time of 10 seconds or less, more preferably 5 seconds or less, most preferably 2 seconds or less.

In a further preferred aspect of the invention, when either the first and/or second mixture are in a solid powder form, they can be dried by heating the powders to reduce the level of liquid material present, without needing to change the physical state of the mixture.

Many substrates such as paper, cardboard, fabrics or textiles have print applied for background colour, branding or packaging information. For examples industrially Offset Printing, Lithography, Digital Printing, Gravure, Screen Printing or Flexography can be used to apply print to paper. All of these methods use roller technology to apply single-colour inks or pigments to the substrate one at a time. Therefore, there needs to be minimal bleed between the layers of colour to prevent smudging and warping of the printed image. The inventive coatings can prevent bleeding of inks or pigments, particularly when used on low density substrate materials such as tissue, blotting or porous paper by providing a less permeable printing surface. The inventive coatings can be used in conjunction with overprint varnishes (OPVs) to give a gloss, satin or matt finish to printing.

In another aspect, the invention relates to a coated substrate obtained by the method described herein.

In a preferred aspect of the invention, the average thickness of the first coating is between 1 and 20 microns, more preferably between 2 and 15 microns. In an alternatively preferred aspect of the invention, the average density of the first coating is between 0.1 and 20 g/m ².

In another preferred aspect of the invention, the average thickness of the second coating is between 1 and 20 microns, more preferably between 2 and 15 microns.

In an alternatively preferred aspect of the invention, the average density of the second coating is between 0.1 and 20 g/m².

In a preferred aspect of the invention, it is possible to observe with microscopy the presence of two distinct coating layers on a substrate, preferably with Scanning Electron Microscopy.

In a preferred aspect of the invention the first dry coating has at least 30% intermolecular beta-sheets, at least 40% intermolecular beta-sheets, at least 50% intermolecular betasheets, at least 60% intermolecular beta-sheets, at least 70% intermolecular beta-sheets, at least 80% intermolecular beta-sheets, or at least 90% intermolecular beta-sheets and the second dry coating has less than 40% intermolecular beta-sheets, less than 30% intermolecular beta-sheets, less than 20% intermolecular beta-sheets, less than 10% intermolecular beta-sheets. In order to investigate the secondary structure of the plantbased protein in the first or the second coating, Fourier-transform infrared (FTIR) analysis is performed. FTIR spectroscopy data are collected using FTIR VERTEX 70 spectrometer (Broker) with a diamond attenuated total reflection (ATR) element. The first or second coating comprising the plant-based protein needs to be in direct contact with the diamond ATR cell. The data is collected using 128 scans at 4 cm^-1 resolution with background subtractions. For the structural analysis of proteins, the spectra are smoothed with a 2^nd order and seven-point window Savitzky-Golay filter and normalized. The second derivative in the Amide I band (1600 - 1700 cm^-1) is calculated from the smoothed data to deconvolve and quantify the secondary and quaternary structural contributions.

A recyclable item prepared with the coated substrate can easily be recycled as the coating can be easily removed or partly removed from the substrate. This is because the coating is not covalently bonded to the substrate, for example paper. In addition, the ease of removing the coating may encourage recycling of the coated substrate within a single recycling stream, such as a paper recycling stream. The methods of recycling the coated substrates include extracting the coating in one or more aqueous extraction medium having pH value sufficient to separate the coatings from the substrate sequentially or in a single step.

The recycling method can further include performing a size reduction process on the coated substrate prior to extracting the coated substrate in the aqueous extraction medium. Size reduction can include pulping, grinding, or any other type of destructive mechanical process to fragment the coated substrate into smaller fragments, in particular to increase surface area exposure at interfacial regions between the substrate and the coating, thereby enhancing contact between the aqueous extraction medium and coating. In general, smaller fragment sizes can promote extraction efficiency.

The coating materials can be washed off from coated substrates, for example from paper pulp during re pulping. For example, both the first and second coating can be removed from the coated paper using an acidic solution, for example 40% v/v acetic acid solution. Alternatively, a first rinse of pulp with a 70-90 vol% ethanol aqueous solution can be used to remove the second coating from the coated paper followed by a second rinse with an aqueous solution of NaOH with pH adjusted to11 to remove the first coating from the paper.

The recycling method can further include separating the substrate from the aqueous extraction medium and recovering and/or reforming the substrate. Separating the substrate from the aqueous extraction medium can be performed by any suitable solid/liquid separation process, for example filtration or decantation to retain the substrate and remove the aqueous extraction medium with the first and second mixture components therein. Optionally, the separation can be followed by one or more washing steps to remove any residual coating material remaining in and/or on the substrate. If the coated material is pulped, ground, or otherwise size-reduced prior to extraction, the resulting substrate fragments can be recovered after separation from the aqueous extraction medium and then re-formed into a new, recycled substrate, for example recycled paper or other cellulosic substrate. In embodiments, the recovered or reformed porous substrate is substantially free from the coating, for example having 5 wt.% or less coating material remaining relative to the initial coating material prior to extraction. For example, the recovered or reformed substrate can have up to 1 , 2, or 5 wt.% less coating material relative to the initial coating material prior to extraction. Alternatively, or additionally, the recovered or reformed substrate can have 0.1 wt.% or less coating material relative to the substrate material. For example, the recovered or reformed substrate material can have at least up to 0.001 , 0.01 , or 0.1 wt.% less coating material, relative to the material.

Extraction can remove substantially all of the coating from the substrate, for example at least 95, 98, or 99 wt.% and/or up to 90, 95, 98, 99, or 100 wt.% of the coating initially present on the coated substrate.

Alternatively, or additionally, if the pulp is not washed or extracted or partially washed/extracted, the remaining coating materials can become part of the recycled paper.

An adhesive is a substance that is capable of holding materials together in a functional manner by surface attachment that resists separation. Adhesives are routinely used in the manufacture of items for example boxes from cardboard, sachets from paper.

The coatings of the invention can heat seal and therefore act as an adhesive in the preparation of items from coated materials. This negates the need for a separate adhesive which is often non-biodegradable.

Alternatively, if additional sealing is required the coatings of the present invention are compatible with typical adhesives such as starch-based adhesives, polyvinyl acetate-based adhesives, and polyethylene oxide-based adhesives or water dispersible adhesives including thermoplastic elastomer-based adhesives and polyvinyl acetate-based adhesives.

Additionally, adhesives that can dissolve in water during the re-pulping step or the disintegration step of the paper recycling process may be particularly suitable for the items of the present invention.

For items of the present invention that are intentionally left in the environment, for example seeds, or inadvertently are disposed of in the environment, for example paper packaging, it is important they are biodegradable and do not add to the growing micro plastic pollution crisis.

Preferred coatings of the present invention are highly biodegradable leaving little trace of their previous existence. Coatings can be prepared for biodegradation assessment by drying a first coating followed by a second coating according to the methods described herein onto a substrate where they can be scraped off mechanically or peeled off, such as mylar or glass.

In preferred coatings of the present invention, the biodegradation percentage based upon O2 consumption of the coating as measured according to ISO-14851 after 28 days is 70 to 100%, more preferably 80 to 100%, most preferably 85 to 100%.

In preferred coatings of the present invention, the biodegradation percentage based upon CO2 production of the coating as measured according to ISO-14851 after 28 days is 70 to 100%, more preferably 75 to 100%, most preferably 80 to 100%.

In another aspect, the invention relates to a coated substrate comprising on at least part of at least a first surface of the substrate a first coating comprising one or more globulin proteins and on top of at least part of the first coating a second coating comprising one or more prolamin proteins.

The invention also relates to the use of the coated substrate described herein for making an item selected from the group consisting of plates, cups, containers, boxes, cartons, corrugated boxes, wrappers and sachets.

In another aspect an item intended for wrapping or enclosing a product, for example a food box or sachet, may be sealed by heat sealing, wherein the coating itself behaves as an adhesive. For heat sealing to occur, the coatings at the interface must be at or above the thermal onset temperature. Controlling the thicknesses of the coating and substrate allows sufficient heat to be transferred to the interface in the time available. A coating with an external surface comprising prolamin plant-based proteins can produce a seal when heated, therefore not requiring additional adhesive material - a self-sealing coating. In preferred embodiments of the coating when heat-sealed it may be present on one or both sides of the seal prior to applying heat and/or pressure. Therefore, it is possible to seal a coated substrate to an uncoated substrate or a coated substrate to another coated substrate.

Preferably, the coating of the present invention has a heat sealing strength of at least 20 N/m, more preferably at least 40 N/m, more preferably at least 60 N/m, even more preferably at least 80 N/m, even more preferably at least 100 N/m, most preferably at least 120 N/m, as measured by ASTM F88/F88M-15 at 55% relative humidity and 20 °C after the coated item has been conditioned at 55 % relative humidity and at 20 °C for at least one hour and then sealed at a temperature of 120 °C and a pressure of 3 bar applied for a time of 1 second.

In a further aspect, the invention relates to a kit for coating a substrate comprising: a. a first mixture comprising one or more liquids and one or more plant-based globulin proteins; b. a second mixture comprising one or more liquids and one or more plantbased prolamin proteins.

In a preferred kit for coating a substrate of the invention, the first mixture comprises the one or more plant-based globulin proteins in an amount of 5 weight % or more.

In another preferred kit for coating a substrate of the invention, the first mixture comprises one or more proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, sunflower protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably selected from pea protein, potato protein, rapeseed protein, and/or sunflower protein, most preferably pea protein.

In another preferred kit for coating a substrate of the invention, the first mixture additionally comprises one or more plant-based albumin proteins or more or more glutelin proteins.

In another preferred kit for coating a substrate of the invention, the second mixture comprises the one or more plant-based prolamin proteins in an amount of 5 weight % or more.

In another preferred kit for coating a substrate of the invention, the second mixture comprises one or more proteins selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin, most preferably zein protein.

In another preferred kit for coating a substrate of the invention, the second mixture additionally comprises one or more plant-based glutelin proteins. In another preferred kit for coating a substrate of the invention, the first mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids.

In another preferred kit for coating a substrate of the invention, the first mixture comprises glycerol.

In another preferred kit for coating a substrate of the invention, the first mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, p- hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid and carnitine.

In another preferred kit for coating a substrate of the invention, the first mixture comprises acetic acid.

In another preferred kit for coating a substrate of the invention, the first mixture comprises lactic acid.

In another preferred kit for coating a substrate of the invention, the second mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids.

In another preferred kit for coating a substrate of the invention, the second mixture comprises glycerol.

In another preferred kit for coating a substrate of the invention, the second mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, p- hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid, and carnitine.

In another preferred kit for coating a substrate of the invention, one of the one or more liquids in the first mixture is water. In another preferred kit for coating a substrate of the invention, one of the one or more liquids in the second mixture is an alcohol.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows boxes made from uncoated and coated card containing vegetable oil.

Figure 2a shows first mixture A moderate shear processing.

Figure 2b shows first mixture N after high shear processing.

EXAMPLES

Ingredients and Materials

Pea Protein Isolate (PPI) (80 wt% protein, 4 wt% carbohydrate) (ProEarth P16109) was purchased from Cambridge Commodities Ltd.

Zein, purified (88-96% protein) was purchased from Fisher Scientific.

Soy Protein Isolate (SPI) was purchased from Fytomax Nutrition Pvt. Ltd.

Food-grade glycerol (APC Pure) was purchased from APC.

Acetic acid (glacial, food grade) was purchased from Fisher Scientific.

Lactic acid (85% solids) was purchased from Sigma Aldrich.

HCI Fisher Chemical Hydrochloric Acid Solution 1 M (1 N), NIST Standard Solution was purchased from Fisher Scientific.

210 gsm A3 White Matt Paper Card Sheets, Product code: 976434 was purchased from The Range, UK.

Ethanol Absolute 99.8+% was purchased from Fisher Scientific.

Castor oil, Thermo Scientific Chemicals was purchased from Fisher Scientific.

Toluene, 99.85%, Extra Dry over Molecular Sieve, AcroSeal™, Thermo Scientific Chemicals was purchased from Fisher Scientific. n-Heptane, 99%, Thermo Scientific Chemicals was purchased from Fisher Scientific.

Vegetable oil was purchased from Tesco UK Pic local supermarket.

8M urea was purchased from Fisher Scientific.

3M thiourea was purchased from Fisher Scientific. sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) was purchased from BIO-RAD, UK. tris/glycine/SDS buffer (25mM Tris, 192 mM glycine and 0.1% w/v SDS) was purchased from Fisher Scientific. Sodium benzoate was purchased from Fisher Scientific.

In the Examples that follow, all references to “ambient temperature” are to a temperature of approximately 20°C. In addition, the weight percentages of materials used in the preparation of the examples refer to the materials as purchased and do not take account of their purity.

Measurement methods

Kit test

Following a TAPPI standard T 559 (Technical Association of the Paper and Pulp Industry), test solutions comprising of castor oil, toluene and n-heptane were blended as follows:

A sample of test paper specimen was selected and conditioned for a minimum of 24 hours at 50% relative humidity and 23°C. Under extraction in a fume cupboard, a drop of test solution 6 was added to the paper. After 15 seconds, excess test solution was wiped off and the sample examined for dark patches which show coating failure. This was repeated 5 times for each test solution. If the sample shows no dark patches it passes, the solution with the higher number is tested until samples fail, or the highest number test solution is reached. If the sample fails, the solution with a lower number is then tested until samples pass, or the lowest number test solution is reached. Once the highest value test solution that does not cause failure is identified, then this is the kit rating of the specimen.

The lowest rating is 0 and indicates the material has no oil resistance under these test conditions. The maximum rating is 12 indicating the material has very good oil resistance under these test conditions.

Cobb test

Cobb 60 values were determined by following a TAPPI standard T441 om-09 (Technical Association of the Paper and Pulp Industry). 125x125mm samples were cut from a test paper specimen and equilibrated for a minimum of 24h at 50% RH and 23°C. Once equilibrated, samples were weighted and then placed in a 11 .3 mm diameter Cobb tester (CT/100) and clamped. 100ml of water was added for 60 seconds, then removed, blotted with blotting paper and a 10kg roller and then re-weighed. The Cobb value was calculated as follows:

Weight of water, g/m² = [Final weight, g - Conditioned weight, g] x 100

Dry Paper GSM

125x125mm samples were cut from paper specimens, equilibrated for a minimum of 24h at 50% RH and 23°C and weighed to 0.001g. The GSM was calculated as follows:

GSM = Weight, g x 10,000 / [length, cm x Width, cm]

Dry Coating GSM

The same procedure as above was used as for Dry Paper GSM after it was coated. The Coating GSM was calculated as the Coated Paper GSM minus the Uncoated Paper GSM.

Dry Paper Thickness

125x125mm samples were cut from paper specimens, equilibrated for a minimum of 24h at 50% RH and 23°C and thickness was measured using a micrometre accurate to 0.001 mm. 5 values were taken and averaged to give a sample thickness. The paper specimen may be coated or uncoated.

Coating thickness

The Total Coating thickness was calculated as the Dry Total Coated Paper thickness minus the Dry Uncoated Paper thickness with the paper conditioned and the thickness measured as above. This can be undertaken for the paper after only the first coating has been applied and dried to obtain the Dry First Coating thickness. To obtain the Dry Second Coating thickness the Dry First Coating thickness is subtracted from the Total Coating thickness.

Method to identify of qlobulins and

A 5x5cm sample of coated material was first placed in a beaker containing 25ml of an 80vol% ethanol aqueous solution. The sample was then placed in an Ultrasonic bath (Fisherbrand FB15051) on maximum setting for 30min to dissolve any prolamin protein fractions. The 80vol% ethanol aqueous solution was filtered using a 0.22pm filter and concentrated to dryness using a freeze-dryer. The recovered solid pellet was re-suspended in an aqueous 8M urea/3M thiourea solution and loaded into a sodium dodecyl sulphatepolyacrylamide gel (SDS-PAGE) using Mini-PROTEAN TGX 4 -15% gels with tris/glycine/SDS buffer for gel electrophoresis analysis. As a control reference, a standard 80% ethanol aqueous solution containing an isolated prolamin protein (e.g. alpha-zein), prepared following the method described above, was loaded into the polyacrylamide gel (SDS-PAGE). The presence of prolamin proteins was confirmed by comparison of the test sample with the control.

A 5x5cm sample of coated material was first placed in a beaker containing 25ml of aqueous solution adjusted to pH=11 using NaOH. The sample was then placed in an Ultrasonic bath (Fisherbrand FB15051) on maximum setting for 30min to dissolve any globulin protein fractions. The alkaline aqueous solution was filtered using a 0.22pm filter and concentrated to dryness using a freeze-dryer. The recovered solid pellet was re-suspended in an aqueous 8M urea/3M thiourea solution and loaded into a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using Mini-PROTEAN TGX 4 -15% gels with tris/glycine/SDS buffer for gel electrophoresis analysis. As a control reference, a standard aqueous solution containing a globulin-rich plant protein (e.g. Pea Protein Isolate), prepared following the method described above, was loaded into the polyacrylamide gel (SDS-PAGE). The presence of prolamin proteins is confirmed by comparison of the test sample with the control.

Viscosity measurements of the mixtures were made using an Anton Paar MCR 92 Rheometer using a plate and cone measurement geometry with a 50mm plate and 1 degree angle. The first mixture was tested on a shear sweep from 0.1 s^-1 to 1000 s^-1, then sheared at 1000 s^-1 for 10 seconds and then swept again at 20°C. The pre and post-shear 50 s^-1 values were reported 20 °C.

Particle size

Particle size measurements of the first mixture were carried out using a laser diffraction technique with an Anton Paar PSA 1190. Measurements were carried out by diluting the mixture in an aqueous solution with acetic acid or lactic acid. It is important that the pH of a sample is away from the isoelectric point of that sample so as to avoid misleading results due to coagulation. For example, Pea Protein Isolate has an isoelectric point of 4.5 and the pH of the mixtures was adjusted to 3 with acetic acid or lactic acid prior to measurement. The slurry was diluted to the required concentration in order to have the desired optical density (normally 2-8% obscuration) for the measurement. The d50 quoted is for the volume distribution. d10 and d5 values for the volume distribution can also be obtained in this way using laser diffraction.

Particle size measurements of the second mixture were carried out using a Dynamic Light Scattering (DLS) technique using a Zeta Sizer Nano S from Malvern P Analytical and operated according to the manufacturer’s instructions. It is important that the mixture is sufficiently diluted so as to avoid misleading results due to particles coagulating during testing. The mixtures were diluted by a factor of 100 with 80% ethanol. 200 DL of the diluted slurry was placed in a cuvette and positioned in the equipment. Testing was then carried out according to the standard equipment procedures. The d50 measured is for the wt/volume distribution. DLS can generally be used to measure particle size up to 500nm. The upper limit is primarily governed by the onset of sedimentation. For larger particle sizes the laser diffraction technique described herein for the first mixture can also be used. mixtures

Coating mixtures A to M were prepared according to the procedures described below.

(i) Preparation of pea protein mixture A

180 g of Reverse Osmosis water was mixed with 19.8 g of Pea Protein Isolate in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C the sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning.

(ii) Preparation of pea protein mixture B

166.5 g of Reverse Osmosis water was mixed with 19.8 g of Pea Protein Isolate in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C, 13.5 g Acetic Acid was added, and the sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning.

(iii) Preparation of pea protein mixture C

180 g of Reverse Osmosis water was mixed with 19.8 g of Pea Protein Isolate and 6.93 g Glycerol in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. 14.18 g of 1 M Hydrochloric acid was added to adjust the pH to 3.5. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning. (iv) Preparation of pea protein mixture D

166.5 g of Reverse Osmosis water was mixed with 19.8 g of Soy Protein in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C, 13.5 g Acetic Acid was added, and sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning.

(v) Preparation of pea protein mixture E

200 g of Reverse Osmosis water was mixed with 22 g of Pea Protein Isolate and 7.7 g Glycerol in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning.

(vi) Preparation of pea protein mixture F

166.5 g of Reverse Osmosis water was mixed with 19.8 g of Pea Protein Isolate and 6.93 g Glycerol in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Once at 80°C, 13.5 g Acetic Acid was added, and sample was heated and stirred for 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until it reached 40°C to prevent the surface of the sample from skinning.

(vii) Preparation of pea protein mixture G

166.5 g of Reverse Osmosis water was mixed with 20 g of Pea Protein Isolate in a 1 L glass beaker at ambient temperature using an overhead stirrer until homogenous. The beaker was then placed in a pre-heated water bath at 90°C and stirred until the batch temperature was 80°C. Separately 48.44 g Zein was dissolved in 13.5 g Acetic Acid, warmed and stirred at 50°C. Once the Pea Protein Isolate mix had reached 80°C, the Acetic Acid and Zein mix was added, and sample was heated and stirred for a further 30 minutes. After 30 minutes the sample was removed from the water bath and mixed with a Silverson mixer for 10 minutes at 7000 RPM. The sample was then stirred with an overhead stirrer in an ice bath until 40°C to prevent the surface of the sample from skinning.

(viii) Preparation of zein mixture H

6 g Water was mixed with 24 g ethanol in a falcon tube using a vortex mixer. 7.5 g Zein was added and mixed until homogeneous.

(ix) Preparation of zein mixture I

6 g Water was mixed with 24 g ethanol and 2.01 g Glycerol in a falcon tube using a vortex mixer. 8 g Zein was added and mixed until homogeneous.

(x) Preparation of zein mixture J

6 g Water was mixed with 24 g ethanol and 1 .5 g Acetic Acid in a falcon tube using a vortex mixer. 7.5 g Zein was added and mixed until homogeneous.

(xi) Preparation of PPI mixture N

836.47 g of Reverse Osmosis water was mixed with 1 g of Sodium benzoate and 99 g of Pea Protein Isolate in a 1 .5 litre metal beaker at ambient temperature using an overhead stirrer until homogenous. 63.53 g of Lactic Acid (85% solids) was then added to the mixture under stirring and mixed until homogenous. The resultant slurry was then mixed under moderate shear with a Silverson mixer at 7000 RPM for 10 minutes. The sheared slurry was then passed through a High-Pressure Homogenizer FPG12805 from Homogenising Systems Ltd, at 100 MPa of pressure which also resulted in an increase in the temperature of the slurry at the nozzle. With 5°C cooling, the outlet temperature of the slurry was then reduced to around 30°C.

The compositions of each of the coating mixtures are shown in Table 1 .

Table 1

The dispersion particle size distribution of the first mixtures were measured using the method herein described. For Example A the d50 was 49.4 microns, for Example B it was 13.5 microns, for Example E 6.3 microns and for Example F 13.0 microns.

The viscosity of the first mixtures were measured using the method herein described. For Example B the pre- and post-Silverson viscosities were 473 MPas and 225 MPas respectively, and for Example F 436 MPas and 188 MPas respectively.

Example 2: Coating of paper card

A first coating mixture and a second coating mixture as given in Table 2a-e were applied to paper card according to the two-step method below. The choice of Kbar determined the wet thickness of each coating.

(i) Preparation of 1^st coating

The material was prepared by tightly taping the paper card to a glass slide to reduce wrinkling. 50 g of a first coating mixture was speed mixed at 2000 RPM for 2 minutes under vacuum to shear and de-gas. Approximately 5 ml per A5 sheet of card was then pipetted on to one end of the test material and the appropriate Kbar from RK Print Coat Instruments was used to coat the sample in a continuous and smooth motion to the desired thickness according to the table below, with any excess being spread off the end of the material.

The sample was then placed in a pre-heated 120°C oven for 10 minutes until dry.

The thickness of the dry coating is depended on the solids content of the coating mixture.

(ii) Preparation of 2^nd coating

Samples were left to cool down to ambient after the first coating was dried. If the sample had wrinkled during the first coating, it was re-taped as flat as possible onto the glass slide. 50 g of a second coating mixture was then speed mixed at 2000 RPM for 2 minutes under vacuum to shear and de-gas. Approximately 5 ml per A5 sheet of card was then pipetted on to one end of the test material and the appropriate Kbar from RK Print Coat Instruments was used to coat the sample in a continuous and smooth motion to the desired thickness according to the table above with any excess being spread off the end of the material. The sample was then placed in a pre-heated 120°C oven for 10 minutes until dry.

The paper samples were then tested according to the Kit test and Cobb 60 test described herein and the results shown in Tables 2a to 2d.

Table 2a: Comparative single coating examples

In Example I an uncoated paper sample was tested and found to have very poor oil resistance with a Kit test value of 0 and it absorbed some water with a Cobb 60 value of 17.8. In Example II and Example II* a paper sample was coated with a single coating of pea protein and organic acid mixture B and N respectively according to the method herein described and found have moderate to poor oil resistance with a Kit test value of 9 and 8 respectively. Without wishing to be bound by theory, it is believed that due to the pea protein being added to an aqueous organic acid solution and subjected to heating and shear, the plant proteins partially unfold, resulting in the exposure of hydrophobic amino acids initially buried within the protein native structure. Once partially unfolded, the organic acid has greater access to protonate amino acid residues, as well as enabling the formation of anion salt bridges that stabilise hydrophobic interactions. Also, upon heating at elevated temperatures, protein-protein non-covalent intermolecular contacts are disrupted. Further, it is believed that the application of mechanical agitation, for example ultrasonication, disrupts large colloidal protein aggregates into smaller ones, as well as disrupting protein intermolecular interactions. Further, it is believed that upon cooling the protein mixture, protein-protein non-covalent intermolecular contacts are enabled, thus promoting the selfassembly of plant protein molecules into inter-connected protein aggregates. Drying the pea protein colloidal suspension of protein aggregates on a cellulosic substrate results in the formation of a dense protein coating with acceptable oil resistance. However, the coating had very poor water resistance, with a Cobb 60 test value of 42.2 which was worse than the uncoated paper sample. Without wishing to be bound by theory It is believed that due to the hydrophilic nature of the globulin protein, water is retained in this coating resulting in a higher Cobb test value.

In Example Ill a paper sample was coated with a single coating of pea protein with inorganic acid mixture C according to the method herein described and found to have very poor oil resistance with a Kit test value of 0. Without wishing to be bound by theory it is believed that when the pea protein is added to an aqueous inorganic acid solution and subjected to heating and shear, the plant proteins do not completely unfold, resulting in a colloidal suspension of pea protein particles with a lower degree of protein-protein non-covalent intermolecular interactions when compared to Mixture B. Upon drying, the colloidal suspension of pea protein particles forms a coating which is not fully homogeneous and therefore allows oil to leak through. In addition, the coating had very poor water resistance, with a Cobb 60 test value of 35.5, worse than the uncoated sample.

In Example IV a paper sample was coated with a single coating of zein mixture H according to the method herein described and found that it still had very poor oil resistance with a Kit test value of 0. In addition, the coating was found to have very poor water resistance, with a Cobb 60 test value of 45.4, similar to Example II and worse than the uncoated sample. Without wishing to be bound by theory it is believed that the zein ethanol solution permeates through the paper very quickly and is unable to form an even and defect free coating.

In Example V a paper sample was coated with a single coating of a blend of pea protein with organic acid and zein mixture G according to the method herein described and found to have very good oil resistance with a Kit test value of 12. However, the coating was found to have very poor water resistance, with a Cobb 60 test value of 44.7, similar to Example II. Without wishing to be bound by theory, it is believed that this low level of zein enhances a little the oil barrier properties of Example II but it not sufficient to alter the hydrophilic nature of the globulin pea resulting in a high Cobb test value.

It can therefore be seen that a single protein coating is unable to provide both the oil and water barrier properties required for a versatile coated paper.

Table 2b: Dual coatings examples

In Examples VI, VII and VIII paper samples were coated with two coatings: a first coating of pea protein of mixtures A, B or C and a second coating of zein coating mixture H according to the method herein described. All of the coated paper samples had very good oil resistance with a Kit test value of 12. In addition, the coatings had good water resistance, with a Cobb 60 test value of 8.3 for the pea protein mixture A lower than for the uncoated paper, 20.0 for the pea protein with organic acid mixture B similar to the uncoated paper and 15.3 for the pea protein with inorganic acid mixture C lower than for the uncoated paper. Without wishing to be bound by theory, it is believed that the first globulin protein coating provides good oil resistance, as well as being a suitable surface for good spreading of the second prolamin mixture. The resulting even and defect free second prolamin coating provides good water resistance.

In Example IX a paper sample was coated with two coatings: a first coating of soy protein with organic acid of mixture D and a second coating of zein coating mixture H according to the method herein described. The coated paper sample again had moderate oil resistance with a Kit test value of 10. In addition, the coating was found to also have good water resistance, with a Cobb 60 test value of 20.7 similar to the uncoated paper sample.

By contrast, in Example X, the coatings were applied in the reverse order, with a first coating of zein mixture H followed by a second coating of pea protein mixture B. The oil resistance was poor with a Kit test rating of 8, lower than for Example VII, and a much lower water resistance with a Cobb 60 test rating of 41 .8, lower than for Example VII. This demonstrated the importance of applying the coating mixtures in the correct order as specified in the method.

Table 2c: Dual thin coating examples. Cobb 60 tests done in triplicate

In Example XI a paper sample was coated also with a very thin first coating of pea protein of mixture B and a second coating of zein mixture H according to the method herein described. The coated paper sample had very good oil resistance with a Kit test value of 11 .5. In addition, the coating had moderate water resistance, with a Cobb 60 test value of 15.4, lower than for the uncoated paper.

In Example XII a paper sample was also coated with a very thin coating of both mixtures: a thin coating of pea protein of mixture B and a second thin coating of zein mixture H according to the method herein described. The coated paper sample had moderate oil resistance with a Kit test value of 9. The water resistance, with a Cobb 60 test value of 24.8, was a bit higher than for uncoated paper and much lower than for single layer protein coatings of Examples II to V. This demonstrated that good oil and water resistance was achieved with very thin coatings which have the advantage of minimising the use of materials and their cost, as well as the amount of solvent evaporated and associated processing costs. In particular minimising the thickness of the zein coating reduced the use of undesirable non-aqueous solvents that need recycling and minimise the potential issues of any yellow colouration to the coating.

This demonstrates that it is possible to achieve both good oil and water resistance with two very thin coatings.

Table 2d: Dual coatings examples comprising plasticiser

In Examples XIII to XV paper sample was coated with a first coating of pea protein of mixtures E or F that also contained glycerol and a second coating of zein mixture H according to the method herein described. The coated paper samples had very good oil resistance with Kit test values of 11 and 12 and good to moderate water resistance, with a Cobb 60 test value ranging from 6.4 to 15.3. Without wishing to be bound by theory it is believed that the addition of glycerol to the first coating results in a more flexible coating and provides a superior substrate for the formation of the second coating.

In Example XVI and ZZ another paper sample was coated with a first coating of pea protein of mixture F or N, and a second coating of zein mixture I, that contained glycerol, according to the method herein described. The coated paper samples also had very good oil resistance with a Kit test value of 12 and had moderate water resistance a bit worse than uncoated paper with Cobb 60 test values of 23.5 and 18.1 , respectively.

Table 2e: Dual coating zein solvent examples

In Example XVII a paper sample was coated with a first coating of pea protein of mixture F and a second coating of zein mixture M according to the method herein described. The coated paper samples had very good oil resistance with a Kit test value of 12 but poor water resistance, with a Cobb 60 test value of 25.0, worse than that of uncoated paper.

Example 3: Heat sealing of coated paper

Test samples of width 25 mm were cut to the dimensions given in ASTM F88/F88M-15 and conditioned overnight at 55% relative humidity and 20°C. Test strip samples were then sealed using an RDM heat sealer to give a fin seal. Sealed test specimens were tested using technique A (unsupported) in a Tinnius Olsen tensile tester. A sealing temperature of 130 °C and a dwell time of 1 second and pressure of 4 bar were employed. The maximum force encountered as each specimen was stressed to failure is reported as Newtons/meter (N/m).

The coated card samples XV had a good heat sealing maximum force/width of 134.8 N/m with the paper failing indicating that the seal was stronger than the material itself. Example 4: Use as a box for containing food

The uncoated card of Example I and the coated card of Example XV, with first coating of first mixture F and a second coating of second mixture H, were cut by hand into 170mm by 170mm squares. Each square was folded by hand to form a box shape and stapled at the corners. Figure 1 shows the box of Example I on the left and Example XV on the right.

The test boxes were placed on an absorbent towel and approximately 50 mis of vegetable oil was gently poured into each box. The boxes were observed initially, at time zero, and as shown in the photo of Figure 1 the oil has already begun to seep into the uncoated card of Example I and stain it. The box of Example XV is unaffected by the presence of the oil. The boxes were left at ambient and observed again after 30 minutes. As shown in the photo in Figure 1 the uncoated card of Example I on the left is stained with the oil. If the box is removed from the absorbent paper it is observed that the oil has gone through the uncoated card and onto the absorbent paper below. After 30 minutes the coated card box of Example XV only shows a small amount of oil staining at the creases of the folded card. When the box is removed from the absorbent paper it is observed that there has been no leakage of oil.

Example 5: Effect of shear on first mixture

First mixture A and first mixture N, according to the compositions in Table 1 , were each prepared and processed by methods with different degrees of shear: low, moderate and high.

First mixture A was prepared by mixing 2000 g of Reverse Osmosis water with 220 g of Pea Protein Isolate in a 3 litre metal beaker at ambient temperature using an overhead stirrer until homogenous. The slurry was then mixed with a Silverson mixer for 10 minutes at 7000 RPM at ambient temperature reaching around 22°C. The slurry was then processed using a High Pressure Homogenizer FPG12805 from Homogenising Systems Ltd, at 100 MPa of pressure which also resulted in an increase in the temperature of the slurry at the nozzle. With 5°C cooling, the outlet temperature of the slurry was then reduced to around 30°C.

First mixture N was prepared according to the method in Example 1 (xi). In both cases a first “low shear” sample was taken after all the materials were added and stirred with the overhead stirrer. A second “moderate shear” sample was taken after mixing with the Silverson. A third “high shear” sample was taken after passing through the High Pressure Homogenizer.

The viscosity of the slurry samples and particle size distribution of the protein aggregates were measured according to the methods described herein, and are shown in Table 3:

Table 3

Moderate shear significantly reduced the slurry viscosity. High pressure did not further reduce viscosity. Lower slurry viscosities are advantageous as they make pumping and spraying easier and less energy intensive.

Increasing the level of shear that the slurry was exposed to reduce the protein aggregates average particle size. This is advantageous as it allows for easier suspension of the protein aggregates and more even coating of the substrate. The combination of organic acid in the formulation and high shear resulted in the smallest protein aggregate average particle size. As demonstrated in comparison with Example A (i), increased heat for the same level of shear also results in a reduction of the average particle size.

The slurries were left in glass bottles overnight at 5°C in the fridge and observed in the morning. First mixture A treated with low or moderate shear had visibly separated, whilst first mixture A treated with high shear looked homogeneous. First mixture N treated with low shear had visibly separated, whilst first mixture N treated with moderate or high shear looked homogeneous. Figure 2a shows first mixture A treated with moderate shear has visibly separated. Figure 2b shows first mixture N treated with high shear was homogeneous. The degree of separation was quantified by stirring the samples with an overhead stirrer for 15 minutes and then centrifuging 40ml in a 50ml flacon tube at 5000 rpm for 2 minutes and observing how much clear supernatant was formed. For first mixture A, 22mls of supernatant was obtained after low shear treatment. 27mls of supernatant was obtained after moderate shear treatment. For high shear treatment no clear supernatant was obtained.

For first mixture N, 24mls of supernatant was obtained after low and moderate shear treatment. For high shear treatment no clear supernatant was obtained.

This demonstrated that increased shear, in particular high-pressure homogenization, resulted in a slurry which was physically more stable and therefore easier to process. It had less tendency to separate, if left to wait prior to a coating process, and therefore would not need additional processing to be resuspended into a homogeneous mixture.

Claims

CLAIMS:

1 . A method of coating a substrate comprising the following steps: a. Preparing a first mixture comprising one or more liquids and one or more plantbased globulin proteins; b. Preparing a second mixture comprising one or more liquids and one or more plant-based prolamin proteins; c. Applying the first mixture to at least part of at least a first surface of a substrate to give a first coating; d. Optionally drying the first coating; e. Applying the second mixture on top of at least part of the first coating of the material to give a second coating; f. Drying the first coating and/or the second coating.

2. The method of claim 1, wherein the substrate comprises a fibre-based material.

3. The method of claim 2, wherein the material is a cellulosic material.

4. The method of claim 2 or claim 3, wherein the material is selected from the group consisting of wood, wood pulp, cotton fibres, hemp fibres, jute fibres, sisal fibres, flax fibres, cellulose-based fibres, silica-based fibres, fruits, vegetables, and seeds.

5. The method of any of claims 1 to 4, wherein the substrate is selected from the group consisting of paper, cardboard, corrugated board.

6. The method of any of claims 1 to 5, wherein one of the one or more liquids in the first mixture is water.

7. The method of any of claims 1 to 6, wherein one of the one or more liquids in the second mixture is an alcohol.

8. The method of any of claims 1 to 7, wherein the first mixture comprises the one or more plant-based globulin proteins in an amount of 5 weight % or more.

9. The method of any of claims 1 to 8, wherein the first mixture comprises one or more proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, sunflower protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably selected from pea protein, potato protein, rapeseed protein, and/or sunflower protein.

10. The method of any of claims 1 to 9, wherein the first mixture comprises pea protein.

11. The method of any of claims 1 to 10, wherein the first mixture additionally comprises one or more plant-based albumin proteins.

12. The method of any of claims 1 to 11, wherein the second mixture comprises the one or more plant-based prolamin proteins in an amount of 5 weight % or more.

13. The method of any of claims 1 to 12, wherein the second mixture comprises one or more proteins selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin.

14. The method of any of claims 1 to 13, wherein the second mixture comprises zein.

15. The method of any of claims 1 to 14, wherein the second mixture additionally comprises one or more plant-based glutelin proteins.

16. The method of any of claims 1 to 15, wherein the first mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids, preferably wherein the first mixture comprises glycerol.

17. The method of claim any if claims 1 to 16, wherein the first and/or second mixture additionally comprises one or more biodegradable polymers selected from starches, algae derived polysaccharides, fungi derived polysaccharides, microbial derived polysaccharides, microbial derived polyesters, chitosan, lignin and cellulose.

18. The method of any of claims 1 to 17, wherein the first mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, p- hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid and carnitine.

19. The method of claim 18, wherein the first mixture comprises acetic acid and/or lactic acid, preferably wherein the first mixture comprises lactic acid.

20. The method of any of claims 1 to 19, wherein the second mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids, preferably wherein the second mixture further comprises glycerol.

21. The method of any of claims 1 to 20, wherein the second mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, p- hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p-methylbutyric acid, 2- hydroxybenzoic acid, and carnitine.

22. The method of any of claims 1 to 21 , wherein the first mixture is prepared by applying high shear using a high pressure homogeniser or sonicator.

23. The method of any of claims 1 to 22, wherein applying the first and/or second mixture to the material is achieved by roller coating, drum coating, rotary coating, dip coating, slot dies, air knives, spray coating, fluid bed coating or extruding.

24. The method of any of claims 1 to 23, wherein drying of the first coating and/or the second coating is achieved by non-contact drying, preferably by using a fan.

25. The method of claim 24, wherein when the substrate is paper or cardboard, drying of the first coating and/or the second coating is achieved by exposure to elevated temperatures for a period of time of 10 seconds or less, more preferably 5 seconds or less, most preferably 2 seconds or less.

26. A coated material obtained by the method of any of claims 1 to 25.

27. The coated material of claim 26, wherein the average thickness of the first coating is between 1 and 20 microns.

28. The coated material of claim 26 or claim 27, wherein the average weight of the first coating is between 0.1 and 20 g/m².

29. The coated material of any of claims 26 to 28, wherein the average thickness of the second coating is between 1 and 20 microns.

30. The coated material of any of claims 26 to 29, wherein the average weight of the second coating is between 0.1 and 20 g/m².

31. The coated material of any of claims 26 to 30, wherein the biodegradation percentage based upon O2 consumption of the coating as measured according to ISO-14851 after 28 days is 70 to 100%, or wherein the biodegradation percentage based upon CO2 production of the film as measured according to ISO-14851 after 28 days is 70 to 100%.

32. The coated material of any of claims 26 to 31 , wherein the coated material consists of food grade ingredients.

33. A coated material comprising on at least part of at least a first surface of the substrate a first coating comprising one or more plant-based globulin proteins and on top of at least part of the first coating a second coating comprising one or more plant-based prolamin proteins.

34. A kit for coating a substrate comprising: a. a first mixture comprising one or more liquids and one or more plant-based globulin proteins; b. a second mixture comprising one or more liquids and one or more plantbased prolamin proteins.

35. The kit for coating a substrate of claim 34, wherein the first mixture comprises the one or more plant-based globulin proteins in an amount of 5 weight % or more.

36. The kit for coating a substrate of claim 34 or claim 35, wherein the first mixture comprises one or more proteins selected from the group consisting of soybean protein, pea protein, rice protein, potato protein, rapeseed protein, sunflower protein, lentil protein, chickpea protein, bean protein, fava bean protein, mung bean protein, sunflower seed protein, pumpkin seed protein, flax protein, chia protein, canola protein, lupine protein, alfalfa protein, moringa protein, borage protein, hemp seed protein and cotton seed protein, preferably selected from pea protein, potato protein, rapeseed protein, and/or sunflower protein.

37. The kit for coating a substrate of any of claims 34 to 36, wherein the first mixture comprises pea protein.

38. The kit for coating a substrate of any of claims 34 to 37, wherein the first mixture additionally comprises one or more plant-based albumin proteins.

39. The kit for coating a substrate of any of claims 34 to 38, wherein the second mixture comprises the one or more plant-based prolamin proteins in an amount of 5 weight % or more.

40. The kit for coating a substrate of any of claims 34 to 39, wherein the second mixture comprises one or more proteins selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin.

41. The kit for coating a substrate of any of claims 34 to 40, wherein the second mixture comprises zein protein.

42. The kit for coating a substrate of any of claims 34 to 41 , wherein the second mixture additionally comprises one or more plant-based glutelin proteins.

43. The kit for coating a substrate of any of claims 34 to 42, wherein the first mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids.

44. The kit for coating a substrate of claim 43, wherein the first mixture comprises glycerol.

45. The kit for coating a substrate of any of claims 34 to 44, wherein the first mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, P-hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p- methylbutyric acid, 2-hydroxybenzoic acid and carnitine.

46. The kit for coating a substrate of claim 45, wherein the first mixture comprises acetic acid and/or lactic acid.

47. The kit for coating a substrate of any of claims 43 to 46, wherein the second mixture further comprises one or more plasticisers selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, sorbitol, mannitol, xylitol, triethyl citrate, monoglycerides, diglycerides, triglyceride glucose, glucose, mannose, fructose, sucrose, urea, lecithin, waxes, and amino acids.

48. The kit for coating a substrate of claim 47, wherein the second mixture comprises glycerol.

49. The kit for coating a substrate of any of claims 34 to 48, wherein the second mixture further comprises one or more organic acids selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycolic acid, gluconic acid, tartaric acid, P-hydroxypropionic acid, p-hydroxybutyric acid, p-hydroxy p- methylbutyric acid, 2-hydroxybenzoic acid, and carnitine.

50. The kit for coating a substrate of any of claims 34 to 49, wherein the first and/or second mixture additionally comprises one or more biodegradable polymers selected from starches, algae derived polysaccharides, fungi derived polysaccharides, microbial derived polysaccharides, microbial derived polyesters, chitosan, lignin and cellulose.

51. The kit for coating a substrate of any of claims 34 to 50, wherein one of the one or more liquids in the first mixture is water.

52. The kit for coating a substrate of any of claims 34 to 51 , wherein one of the one or more liquids in the second mixture is an alcohol.

53. Use of the coated substrate of any of claims 26 to 33 for making an item selected from the group consisting of plates, cups, containers, boxes, cartons, corrugated boxes, wrappers and sachets.

54. Use according to claim 53, wherein the item is self-heat-sealing.