A MODIFIED GUM

FIELD OF THE INVENTION

This invention relates to a modified gum. In particular, the invention relates to a modified polysaccharide or gum for use in preparing a food thickening composition and method of making same. The invention further relates to a stable liquid composition containing the modified gum and a thickening agent for increasing the viscosity of a liquid or semi-liquid foodstuff and a method of using same.

BACKGROUND TO THE INVENTION

It is often desirable to provide viscous thickened liquids, particularly for the geriatric and convalescent markets. The thickened liquids need to be of a particular, known and repeatable viscosity to be applicable to these markets. Thickening beverages for the management of dysphagia in institutions and homes is typically achieved using powdered or liquid thickeners.

The use of commercially available liquid thickeners that function by way of expressing the viscosity of a thickening agent in a concentrated solution and diluting back to a desired concentration is becoming more widespread. Their use, however, may produce an undesirable haziness or cloudiness in clear drinks, such as water, when thickened with such liquid thickeners.

Thus, there remains a need for a viscosity-inhibited liquid thickener composition that may be used, for example, to feed subjects suffering from a mastication and/or deglutition disorder, such as dysphagia, which overcomes or ameliorates this limitation of commercially available liquid thickener compositions.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a modified gum for use in preparing a liquid thickening composition comprising: a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof; wherein the gum-based source material has been treated with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease.

In one particular embodiment, the gum-based source material has been treated with: (a) a proline protease and/or a serine protease; and (b) a cysteine protease.

In a second aspect, the invention resides in a method of preparing a modified gum for use in preparing a liquid thickening composition including the steps of:

(i) providing a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof;

(ii) treating the gum-based source material with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease; to thereby prepare the modified gum.

In one particular embodiment, step (ii) of the method includes treating the gum- based source material with: (a) a proline protease and/or a serine protease; and (b) a cysteine protease.

Referring to the aforementioned aspects, the proline protease is suitably selected from the group consisting of a proline-specific endopeptidase, such as one at least partly derived from an Aspergillus spp, a prolyl oligopeptidase, a dipeptidyl peptidase IV, a prolidase, an aminopeptidase P, a carboxypeptidase P and any combination thereof.

In regard to the invention of the first and second aspects, the cysteine protease may be selected from the group consisting of papain, a papain-like cysteine protease, bromelain, cathepsin K, calpain, caspase, separase and any combination thereof. In specific embodiments, the cysteine protease is or comprises papain.

With respect to the above aspects, the serine protease is suitably selected from the group consisting of a serine-specific endopeptidase, a subtilisin, a prolyl oligopeptidase, trypsin and any combination thereof. In particular embodiments, the serine-specific endopeptidase is at least partly derived from a Bacillus spp., such as Bacillus licheniformis.

In a third aspect, the invention provides a modified gum produced according to the method of the second aspect. In a fourth aspect, the invention resides in a stable liquid composition comprising:

(i) one or a plurality of thickening agents; and

(ii) a modified gum comprising a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof, wherein the gum-based source material has been treated with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease; wherein addition of the composition to an aqueous liquid or aqueous liquid solid mixture foodstuff increases the viscosity of said foodstuff.

In various embodiments, the modified gum is that of the first aspect.

Suitably, the thickening agent is selected from the group consisting of agar, alginic acid, carrageenan, a guar gum, gum tragacanth, gum ghatti, microcrystalline cellulose, sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylethylcellulose, gum karaya, a xanthan gum, a locust bean gum, a tara gum, a psyllium seed gum, a quince seed gum, a pectin, furcellaran, a gellan gum, konjac, sodium alginate and any combination thereof.

In some embodiments, the thickening agent is a xanthan gum. For such embodiments, the xanthan gum suitably has a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5.

In a fifth aspect, the invention resides in a stable liquid composition comprising:

(i) one or a plurality of thickening agents, the thickening agents comprising a xanthan gum having a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5; and

(ii) a gum, the gum optionally selected from the group consisting of a bean extract, a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof; wherein addition of the composition to an aqueous liquid or aqueous liquid solid mixture foodstuff increases the viscosity of said foodstuff. In certain embodiments of the fourth and fifth aspects, the xanthan gum is derived at least in part from a Xanthomonas campestris strain selected from the group consisting of pathovar cynarae CFBP 19, juglandis CFBP 176, pelargonii CFBP 64, phaseoli CFBP 412 or ATCC 17915, celebenois ATCC 19046, corylina CFBP 1847, or from a derivative, variant or progeny thereof, and any combination thereof.

In various embodiments of the fourth and fifth aspects, the gum or gum -based source material is selected from the group consisting of a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof.

In some embodiments of the fourth and fifth aspects, the composition has a viscosity of less than 4000 cP or less than 2000 cP.

In various embodiments of the fourth and fifth aspects, the composition has a water activity of greater than 95%.

In particular embodiments of the two aforementioned aspects, the composition is stable for at least six months at room temperature.

In certain embodiments of the fourth and fifth aspects, the stable liquid composition has a pH of about 3.5 to about 4.5.

In one embodiment of the two previous aspects, the stable liquid composition comprises potassium chloride at a concentration of about 0.01 mol/L to about 0.5 mol/L.

In other embodiments of the fourth and fifth aspects, the stable liquid composition of the present aspect has a Brix value of about 20° Bx to about 30° Bx.

In a sixth aspect, the invention resides in a method of producing a stable liquid composition, including the steps of:

(i) providing a modified gum according to the first or third aspects;

(ii) adding one or a plurality of thickening agents to the modified gum; and

(iii) mixing the mixture of step (ii) to thereby produce the stable liquid composition.

Suitably, the stable liquid composition is that of the fourth aspect.

In a seventh aspect, the invention provides a method of producing a stable liquid composition, including the steps of: (i) providing one or a plurality of thickening agents, the thickening agents comprising a xanthan gum having a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5; and

(ii) adding a gum, the gum optionally selected from the group consisting of a bean extract, a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof to the thickening agents; and

(iii) mixing the mixture of step (ii) to thereby produce the stable liquid composition.

In various embodiments, the gum is selected from the group consisting of a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof.

Suitably, the stable liquid composition is that of the fifth aspect.

In certain embodiments, the method of the two previous aspects further includes the step of adjusting the pH of the stable liquid composition to a pH of about 3.5 to about 4.5.

In some embodiments, the method of the sixth and seventh aspects further includes the step of adding potassium chloride to a concentration of about 0.01 mol/L to about 0.5 mol/L.

In other embodiments, the method of the two previous aspects further includes the step of adjusting a Brix value of the stable liquid composition to about 20° Bx to about 30° Bx.

In an eighth aspect, the invention provides a stable liquid composition produced according to the method of the sixth or seventh aspects.

In a ninth aspect, the invention provides a method for increasing the viscosity of an aqueous liquid or aqueous liquid solid mixture foodstuff, the method including the steps of:

(a) adding to the foodstuff a stable liquid composition of the fourth, fifth or eighth aspects; and

(b) mixing the foodstuff and the composition so as to promote increasing the viscosity of said foodstuff by the composition.

Suitably, the mixing step includes applying low-shear mixing. In this regard, the low-shear mixing may be applied for about 30 seconds or less to achieve a maximal viscosity of the foodstuff. Alternatively, the low-shear mixing may be applied for about 10 to about 30 seconds to achieve a maximal viscosity of the foodstuff. In certain embodiments, the low-shear mixing comprises stirring said composition at a speed of from about 10 rpm to about 150 rpm.

In one embodiment, the viscosity of said foodstuff is increased to greater than

95 cP.

Suitably, the foodstuff of increased viscosity is for feeding a subject suffering from a mastication and/or deglutition disease, disorder or condition, such as dysphagia.

As used herein, except where the context requires otherwise, the term “ comprise” and variations of the term, such as “comprising", “ comprises” and “comprised”, are not intended to exclude further elements, components, integers or steps but may include one or more unstated further elements, components, integers or steps.

It will be appreciated that the indefinite articles “a” and “an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, “a” polysaccharide includes one polysaccharide, one or more polysaccharides and a plurality of polysaccharides.

As used in this specification, the term “about’ refers to a variation or tolerance in a stated amount, concentration, ratio or proportion, preferably defined as being no more than 10%, 5%, 2% or 1 % above or below a stated amount, concentration, ratio or proportion.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist in understanding the invention and to enable a person skilled in the art to put the invention into practical effect, preferred embodiments of the invention will be described by way of example only with reference to the accompanying drawings.

Figure 1. A) the fully hydrated xanthan solution hydrated in hot water, B) the inhibitory gum rich solution, C) equivalent mixtures of the inhibitory gum rich solution and the fully hydrated xanthan solution prepared hot D) equivalent mixtures of the inhibitory gum rich solution and the fully hydrated xanthan solution prepared cold. E) the fully hydrated xanthan solution hydrated in cold water, F) the inhibitory gum rich solution G) a 5% solution of the viscosity inhibited liquid thickener prepared using hot water (non-treated control) H) a 5% solution of the viscosity inhibited liquid thickener prepared using cold water (non-treated control).

Figure 2. Fibrous insoluble particles visible in the 5% solution of the viscosity inhibited liquid thickener (samples G and FI).

Figure 3. Absorbance (395nm) versus pH relationship of liquid thickener solution diluted in water (5% w/v).

Figure 4. Percent transmitted light at 650nm in 5% (w/v) dilutions of viscosity inhibited liquid thickening agent with increasing concentrations of potassium chloride addition. Figure 5. Visual haze in 5% (w/v) dilutions of viscosity inhibited liquid thickening agent with increasing concentrations of potassium chloride addition.

Figure 6. Correlation between Brix values and absorbance at 395nm.

Figure 7. 4.32% process intermediate solution produced from left to right: trial protein reduced and control variants of inhibitory gum

Figure 8. 5% diluted concentrate solution produced from left to right: protein reduced and control commercial variants of inhibitory gum optimal pH (4.1 ) and total solids (24° Brix).

Figure 9. SDS PAGE of the samples taken during processing; PM indicates lanes containing SeeBlue Plus2 pre-stained protein ladder. Lane 8: pre-proline specific endopeptidase and pre- papain enzyme treated inhibitory gum sol.

Figure 10. 5% liquid thickener solution in water (IDDSI L2) produced from concentrated inhibitory gum solutions treated with papain for (from L-R) 3 hours, 5 hours and 7 hours and untreated control.

Figure 11. Percentage (%) Transmittance (650 nm) of 5% liquid thickener solution in water (IDDSI L2) prepared from intermediate modified gum sequentially treated at ambient temperature with proline specific endopeptidase over 3-16 hours followed by papain treatment; 3 hours at 80°C. Figure 12. Percentage (%) Transmittance (650 nm) of 5% liquid thickener solution in water (IDDSI L2) prepared from enzyme treated inhibitory gum process intermediate with 0.045mol/L KCI sequentially treated at ambient temperature proline specific endopeptidase over 12-18 hours followed by 10 x papain treatment; 3 hours at 80°C. Figure 13. 5% liquid thickener solution in water (IDDSI L2) produced from concentrated modified inhibitory gum solutions treated sequentially with a proline specific endopeptidase and papain (from L-R) Pure xanthan solution, treated inhibitory gum solution, 5 % liquid thickener solution sequentially enzyme treated lower protein gum solution prepared with hot water and 5 % liquid thickener solution sequentially enzyme treated lower protein gum solution prepared with cold water.

Figure 14. 5% liquid thickener solution in water (IDDSI L2) produced from concentrated reduced protein inhibitory gum solutions treated sequentially with a proline specific endopeptidase and papain (from L-R) prepared with hot water and prepared with cold water.

Figure 15. L-R Five percent solutions of the enzyme pre-treated viscosity inhibited liquid thickener produced using A) a xanthan gum having a pyruvic acid content > 5% and a pyruvic acid to acetic acid ratio of > 0.5 B) standard commercially available clear xanthan and C) Five percent solutions of the non- enzyme pre-treated viscosity inhibited liquid thickener produced using commercially available clear xanthan.

Figure 16. Five percent solution of the enzyme pre-treated viscosity inhibited liquid thickener produced using a commercially available low charge density xanthan gum that has been treated by heating for 17 min @ 95°C, cooled, then centrifuged @ 1500 rpm for 10 min.

DETAILED DESCRIPTION OF THE INVENTION The invention advantageously provides a modified gum for use in preparing a viscosity-inhibited liquid thickener that produces less turbidity and improvements in clarity when dispersed and viscosity expressed in clear or semi-clear liquid or semi liquid foodstuffs, such as water. Also, the invention advantageously provides additional means, such as the addition of KCI and pH and/or Brix value adjustments, that can further improve the clarity of clear foodstuffs thickened with a viscosity-inhibited liquid thickener. As such, in one broad form, the invention relates to a modified gum for use in preparing a liquid thickening composition comprising a gum-based source material that has been treated with one or more enzymes or proteases, such as a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease.

Accordingly, in one aspect, the invention provides a modified gum for use in preparing a liquid thickening composition comprising: a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof; wherein the gum-based source material has been treated with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease.

In one embodiment, the gum-based source material has been treated with: (a) a proline protease and/or a serine protease; and (b) a cysteine protease.

In a related aspect, the invention provides a method of preparing a modified gum for use in preparing a liquid thickening composition including the steps of:

(i) providing a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof; and

(ii) treating the gum-based source material with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease; to thereby prepare the modified gum.

In one particular embodiment, step (ii) of the method includes treating the gum- based source material with: (a) a proline protease and/or a serine protease; and (b) a cysteine protease.

The statements which follow apply equally to the aforementioned aspects of the invention.

The modified gum refers to a modified gum-based source material, such as a plant gum, which has been subjected to enzymatic treatment to degrade or modify a specific portion, such as a protein portion and/or a carbohydrate portion, inclusive of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, thereof.

The term “gum”, as used herein, refers to any synthetic polymer, natural polysaccharide, or derivatized natural polysaccharide that is edible and capable of increasing the viscosity of a solution, such as to a semi-solid or gelatinous state, when added thereto. In particular embodiments, the gum-based source material is or comprises one or more natural polysaccharide gums or extracts, such as gum arabic. Examples of natural polysaccharide gums include, but are not limited to, carrageenan, konjac, sodium alginate, aloe vera gel, agarose, guar, pectin, tragacanth, acacia, Arabic, curdlan, gellan, xanthan, scleroglucan, hyaluronic acid, or chitosan. Examples of derivatized natural polysaccharide gums include, but are not limited to, propyleneglycol alginate and hydroxypropyl guar.

The term “polysaccharide", as used herein, generally refers to polymers formed from about 10 to over 100,000 saccharide units linked to each other by hemiacetal or glycosidic bonds. The polysaccharide may be either a straight chain, singly branched, or multiply branched wherein each branch may have additional secondary branches, and the monosaccharides may be standard D- or L-cyclic sugars in the pyranose (6- membered ring) or furanose (5- membered ring) forms such as D-fructose and D- galactose, respectively. Additionally, they may be cyclic sugar derivatives, deoxy sugars, sugar, sugar acids, or multi-derivatized sugars. As would be understood by the skilled artisan, polysaccharide preparations, and in particular those isolated or purified from nature or a natural source, typically comprise molecules that are heterogeneous in molecular weight.

The term "gum-based source material" refers to materials containing one or a plurality of gums, such as those described herein, as a major component thereof (e.g., the gum-based source material comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or any range therein of a gum by weight of the gum-based source material). Accordingly, the gum-based source material may include other components, such as protein, lipid etc, as a minor component thereof.

In various embodiments of invention described herein, the gum or gum-based source material is selected from the group consisting of a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof.

As described herein, the gum-based source material, such as a plant extract or gum described herein, also contains a protein portion as a component thereof. In certain embodiments, the gum-based source material has an initial protein content or level of about or less than about 20 wt% (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5 wt% and any range therein), suitably less than about 10 wt% and more suitably less than about 6 wt% based on the total weight of the gum- based source material.

By “protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids as are well understood in the art. The term “protein” includes and encompasses “peptide”, which is typically used to describe a protein having no more than fifty (50) amino acids and “polypeptide”, which is typically used to describe a protein having more than fifty (50) amino acids.

The term "protease" is defined herein as an enzyme that hydrolyses peptide bonds. The term "protease" can include any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California. As will be appreciated, proteases are classified on the basis of their catalytic mechanism into the following groups: serine endoproteases (EC 3.4.21 ), cysteine endoproteases (EC 3.4.22), aspartic endoproteases (EC 3.4.23), metalloendoproteases (EC 3.4.24) and threonine endoproteases (EC 3.4.25) (see, e.g., Handbook of Proteolytic Enzymes, A.J. Barrett, N.D. Rawlings, J.F.Woessner (eds), Academic Press (1998)).

The proteases used herein can be from, for example, fruit, animal origin, bacteria or fungi. The protease may have endo-activity and/or exo-activity or any combination thereof. It will be understood that suitable proteases for use in the process of the invention are available from commercial suppliers, such as Novozymes, Genencor, AB-Enzymes and DSM Food Specialities Amano, albeit without limitation thereto. Exemplary proteases are those of bacterial or fungal origin, such as from

Bacillus licheniformes or Aspergillus oryzae. The skilled person will appreciate that the amount or concentration of the one or more enzymes (i.e. , a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease) utilised in the treatment of the gum-based source material may depend on, for example, the type of enzyme or enzymes to be utilised, the amount and type of gum-based source material to be treated and the reaction or treatment conditions (e.g., time, temperatures etc) required. In particular embodiments, the one or more enzymes described herein are present in an amount or concentration of about 0.01 % to about 10% (e.g., about 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 % and any range therein) by weight or volume of a reaction mixture containing the one or more enzymes described herein.

The term “proline protease” or "proline-specific protease" refers to an endoprotease or exopeptidase which is capable of cleaving a peptide bond that includes a proline residue. Exemplary proteases capable of cleaving such peptide bonds include a proline-specific endopeptidase, such as one at least partly derived from an Aspergillus spp, a prolyl oligopeptidase and a dipeptidyl peptidase (e.g., dipeptidyl peptidase II and dipeptidyl peptidase IV) a prolidase, an aminopeptidase P and a carboxypeptidase P.

Similarly, the term “serine protease” or “serine-specific protease” is used herein to describe a protease which is capable of cleaving a peptide bond that includes a serine residue. Exemplary proteases capable of cleaving such peptide bonds include serine-specific endopeptidases, subtilisin, trypsin and prolyl oligopeptidases. In particular embodiments, the serine-specific endopeptidase is at least partly derived from a Bacillus spp. , such as Bacillus licheniformis.

Additionally, the term “cysteine protease” or “cysteine-specific protease” is used herein to describe a protease which is capable of cleaving a peptide bond that includes a cysteine residue. Exemplary proteases capable of cleaving such peptide bonds include papain, a papain-like cysteine protease, bromelain, cathepsin K, caspase, separase and calpain. In one particular embodiment, the cysteine protease is or comprises papain.

Referring to the protease treatment steps described herein, protease treatment (inclusive of cysteine proteases, serine proteases, proline proteases, metalloproteases and carbohydrases) is suitably carried out at a pH of about 4.0 to about 9.0, and more particularly about 5.0 to 8.0 or any range therein. In particular embodiments, protease treatment is carried out at a pH of about 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0 and any range therein.

Further to the above, the protease treatment steps described herein (inclusive of cysteine proteases, serine proteases, proline proteases, metalloproteases and carbohydrases) can be carried out at a temperature of about 15°C to about 90°C, and more particularly about 50°C to about 80°C or any range therein. In particular embodiments, protease treatment is carried out at a temperature of about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C and any range therein.

In relation to the aspects described herein, protease treatment (inclusive of cysteine proteases, serine proteases, proline proteases, metalloproteases and carbohydrases) is suitably carried out for a period of time from about 15 minutes to about 48 hours, particularly about 2 hours to about 12 hours and more particularly from about 4 hours to about 18 hours and any range therein. In particular embodiments, protease treatment is carried out for a period of time of about 15 min,

20 min, 30 min, 40 min, 50 min, 1 hr, 1 .25 hr, 1 .5 hr, 1 .75 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr,

7 hr, 8 hr, 9 hr, 10 hr, 11 hr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr, 18 hr, 19 hr, 20 hr,

21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28 hr, 29 hr, 30 hr, 31 hr, 32 hr, 33 hr, 34 hr, 35 hr, 36 hr, 37 hr, 38 hr, 39 hr, 40 hr, 41 hr, 42 hr, 43 hr, 44 hr, 45 hr, 46 hr, 47 hr,

48 hr and any range therein.

In some embodiments, the gum-based source material has been subjected to a protein hydrolysis and/or a protein extraction step (such as that described in PCT/AU2019/050171 , which is incorporated by reference herein) prior to or after treatment with the aforementioned proteases. In this regard, the protein hydrolysis and/or protein extraction steps may have lowered an initial protein level of the gum- based source material to a second protein level. As such, in some embodiments, the second protein content or level produced following treatment of the gum-based source material in step (ii) above is less than about 20 wt% (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5 wt% and any range therein).

In particular embodiments, the protein hydrolysis step produces or results in a degree of hydrolysis of a protein content or fraction of the gum or gum -based source material of about 1 % to about 30% (e.g., about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or any range therein) or more particularly about 5% to about 20%.

By "protein hydrolysis" or “hydrolysing the protein” is meant a process of cleavage or breakage of the chemical bonds that hold the protein material together such that the protein is hydrolyzed or broken down into smaller peptides or protein fragments typically having a reduced molecular weight than the protein in its original (i.e. , unhydrolyzed) state. It will be appreciated that protein hydrolysis may only be partial. "Partial hydrolysis" or "partially hydrolyses" and any grammatical variants thereof, as used herein, refer to the hydrolysis reaction cleaving or breaking less than 100% of the chemical bonds that hold the protein together. By way of example, protein can be hydrolyzed using heat treatment, an acid, a base, one or more further enzymes, or any combination of any of these.

Accordingly, in particular embodiments, the protein hydrolysis step comprises one or more of heat treatment, protease treatment, acid treatment, alkali treatment, microwave radiation treatment, acoustic treatment and metal aqua ion treatment. In various embodiments, the protein hydrolysis step includes heat treatment and/or acid treatment. In this regard, the protein hydrolysis step may include: (a) acid treatment alone; (b) heat treatment alone; (c) sequentially with acid treatment and then heat treatment; or (d) sequentially with heat treatment and then acid treatment.

As used herein, “treating” or “treatment” may refer to, for example, contacting, soaking, steam impregnating, spraying, suspending, immersing, saturating, dipping, wetting, rinsing, washing, submerging, and/or any variation and/or combination thereof.

The skilled person would readily understand that the term "acid", as used herein, refers to various water-soluble compounds with a pH of less than 7 that can be reacted with an alkali to form a salt. Examples of acids can be monoprotic or polyprotic and can comprise one, two, three, or more acid functional groups. Examples of acids include, but are not limited to, mineral acids, Lewis acids, acidic metal salts, organic acids, solid acids, inorganic acids, or any combination thereof. Suitably, acid treatment includes contacting the gum-based source material or modified gum with a food grade acid, such as lactic acid, phosphoric acid, citric acid, malic acid, ascorbic acid, formic acid, fumaric acid, succinic acid, tartaric acid, gluconic acid and any combination thereof. In certain embodiments, the acid, such as the food grade acid, has a concentration of about 0.1 to 5 M, and more suitably of about 0.5 to about 2 M.

In one particular embodiment, the food grade acid is or comprises gluconic acid, such as that derived at least in part from glucono delta-lactone. In this regard, it will be appreciated that glucono delta-lactone typically hydrolyses in aqueous solutions to produce gluconic acid.

Referring to the protein hydrolysis step, acid treatment is suitably carried out at a pH of about 2.0 to about 6.0, or more particularly about 3.0 to 4.0 or any range therein. In particular embodiments, acid treatment is carried out at a pH of about 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 and any range therein. In certain embodiments, acid treatment is carried out at a pH of about 4.0 to 4.4.

As would be readily understood by the skilled artisan, "alkali", as used herein, refers to various water-soluble compounds with a pH of greater than 7 that can be reacted with an acid to form a salt. By way of example, an alkali can include, but is not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide and alkali metal salts such as, but not limited to, sodium carbonate and potassium carbonate.

In particular embodiments, the gum-based source material or modified gum may be treated with one or more acids and/or alkalis in respect of the protein hydrolysis step. For example, the gum-based source material or modified gum may be treated with 1 , 2, 3, 4, 5, or more acids and/or alkalis.

For the protein hydrolysis step, the acid and/or alkali may be present in an amount from about 0.1 % to 15% or any range therein such as, but not limited to, about 0.3% to about 13%, or about 1 % to about 10% by weight of the gum-based source material or modified gum. In particular embodiments of the present invention, an acid and/or an alkali is present in the protein hydrolysis step in an amount of about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%, 6.5%, 6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.5%, 8.75%, 9%, 9.25%, 9.5%, 9.75%, 10%, 10.25%, 10.5%, 10.75%, 11 %, 11.25%, 11.5%, 11.75%, 12%, 12.25%, 12.5%, 12.75%, 13%, 13.25%, 13.5%, 13.75%, 14%, 14.25%, 14.5%, 14.75%, 15% or any range therein, by weight of the gum-based source material or modified gum. In certain embodiments of the present invention, an acid and/or alkali is present in the protein hydrolysis step in an amount of about 1 % to about 2% by weight of the gum-based source material or modified gum .

With respect to the protein hydrolysis step, heat treatment is suitably carried out at a temperature from about 40°C to 99°C, more particularly about 55°C to about 90°C or any range therein, such as, but not limited to, about 65°C to about 85°C or about 45°C to about 80°C. In particular embodiments, heat treatment is carried out at a temperature of about 40°C, 41 °C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71 °C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81 °C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91 °C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C and any range therein. In certain embodiments, heat treatment is carried out at a temperature of about 70°C to about 80°C.

Suitably, the protein hydrolysis step is carried out for a period of time from about

15 minutes to about 48 hours, preferably about 20 minutes to about 12 hours and more particularlyfrom about 30 minutes to about 2 hours and any range therein. In particular embodiments, the protein hydrolysis step is carried out for a period of time of about 15 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 1.25 hr, 1 .5 hr, 1 .75 hr, 2 hr, 3 hr, 4 hr, 5 hr,

6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 11 hr, 12 hr, 13 hr, 14 hr, 15 hr, 16 hr, 17 hr, 18 hr, 19 hr, 20 hr, 21 hr, 22 hr, 23 hr, 24 hr, 25 hr, 26 hr, 27 hr, 28 hr, 29 hr, 30 hr, 31 hr, 32 hr, 33 hr, 34 hr, 35 hr, 36 hr, 37 hr, 38 hr, 39 hr, 40 hr, 41 hr, 42 hr, 43 hr, 44 hr, 45 hr, 46 hr, 47 hr, 48 hr and any range therein.

As generally used herein, the term “protein extraction” refers to the separation, removal and/or isolation of protein and more particularly hydrolysed protein, at least in part, from the gum-based source material, which may be performed by any method or means known in the art. Exemplary methods of protein extraction include centrifugation, size exclusion chromatography, hydrophobic interaction chromatography, gravity separation, ion exchange chromatography, free flow electrophoresis, metal binding, immunoaffinity chromatography and immunoprecipitation.

In some embodiments, the protein extraction step produces a second protein level that is at least about 50%, 40%, 30%, 20%, 15%, 10%, or 5% lower than that of the initial protein level of the gum-based starting material. In particular embodiments, the protein extraction step produces a second protein level that is at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% and any range therein lower than said initial protein level.

With respect to the above, the degree of protein hydrolysis can be determined by any manner known to those skilled in the art (see, e.g., Petersen et al., Determination of the Degree of Hydrolysis (DH) based on OPA Reaction, ED-9512723 Novo Nordisk A/S, Dec. 1995; Frister et al., OPA method modified by use of N,N- dimethyl-2-mercaptoethylammonium chloride as thiol component, Fresenius J. Anal. Chem. 330 (1988) 631 ).

In a further aspect, the invention provides a modified gum or gum-based ingredient prepared by the method of the aforementioned aspect.

Suitably, the modified gum described herein is capable of or is adapted to modulate and/or control the water binding ability of a thickening agent, such as those hereinafter described. To this end, the modified gum may produce specific degrees of viscosity inhibition of a liquid composition, such as those provided herein, comprising the modified gum and a thickening agent. Additionally, the modified gum may further control the rate and extent that their viscosity inhibition is released and/or reversed upon dilution of the liquid composition.

Accordingly, in another aspect, the invention provides a stable liquid composition comprising:

(i) one or a plurality of thickening agents; and

(ii) a modified gum comprising a gum-based source material selected from the group consisting of a bean extract, a Larix occidentalis extract, a Larix laricina extract, an Acacia tree extract, a Larix decidua extract, a Larix sibirica extract and any combination thereof, wherein the gum-based source material has been treated with one or more of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease; wherein addition of the composition to an aqueous liquid or aqueous liquid solid mixture foodstuff increases the viscosity of said foodstuff.

In various embodiments, the modified gum is that of the first mentioned aspect.

It will be appreciated that the term “exoproteases” refers to proteases that hydrolyse peptide bonds adjacent to a terminal a-amino group (“aminopeptidases”), or a peptide bond between the terminal carboxyl group and the penultimate amino acid (“carboxypeptidases”). In particular embodiments, the exoprotease is at least partly derived from an Aspergillus spp., such as Aspergillus oryzae (e.g., Flavourzyme ^® ).

As used herein, the term "metalloprotease", "metalloproteinases" or "metallopeptidase" refers to proteases that require binding of a metal ion, such as zinc, to perform their catalytic activity. It is envisaged that the metalloprotease may be any as are known in the art. In particular embodiments, however, the metalloprotease is or comprises a neutral metalloprotease, such as produced by Bacillus amyloliquefaciens (e.g., metalloprotease sold under the trade name of Neutrase ^® ).

As used herein, the term “carbohydrase” means any enzyme that is capable of at least catalyzing hydrolysis of a carbohydrate-containing target substrate, such as those that contain complex carbohydrates like cellulose, hemicellulose, pectin, xylan, chains of hemicellulose, and/or polymers of other 5-carbon sugars into their sugar components like pentoses or hexoses. The carbohydrase may be any as are known in the art. In particular embodiments, the carbohydrase may include one or more of an arabanase, a cellulase, a b-glucanase, a hemicellulase, and a xylanase, such as those sold under the trade name of Viscoenzyme ^® .

With respect to embodiments in which treatment of the gum-based source material includes treating with more than one of a proline protease, a serine protease, an exopeptidase, a metalloprotease, a carbohydrase and a cysteine protease, it is envisaged that such enzymatic treatment may be performed sequentially and/or simultaneously with respect to the individual enzymes. By way of example, the gum- based source material may be treated: (a) with a proline protease and/or a serine protease first and then a cysteine protease second; (b) with a cysteine protease first and then a proline protease and/or a serine protease second; or (c) simultaneously with a proline protease and/or a serine protease and a cysteine protease.

The term “thickening agent” as used herein refers to those compounds provided herein that are used to increase the viscosity of a liquid mixture and/or solution, and in particular, those for use in food applications, including edible gums, vegetable gums and food-grade polysaccharides. Non-limiting examples of thickening agents include agar, alginic acid, carrageenan, guar gum, gum tragacanth, gum ghatti, microcrystalline cellulose, sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylethylcellulose, gum karaya, xanthan gum, locust bean gum, tara gum, psyllium seed gum, quince seed gum, a pectin, furcellaran, gellan gum, konjac, sodium alginate and any combination thereof.

In various embodiments, the thickening agent is a xanthan gum. In some embodiments, the xanthan gum has a pyruvic acid content of at least about 0.5% (w/w)

(e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,

9.5, 10.0 etc % and any range therein). For some embodiments, the xanthan gum suitably has a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5 (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1 .3, 1 .4, 1 .5, 1.6,

1 .7, 1 .8, 1 .9, 2.0 etc and any range therein), such as those described in US9,380,803.

In certain embodiments, the xanthan gum is derived at least in part from a

Xanthomonas campestris strain selected from the group consisting of pathovar cynarae CFBP 19, juglandis CFBP 176, pelargonii CFBP 64, phaseoli CFBP 412 or

ATCC 17915, celebenois ATCC 19046, corylina CFBP 1847, or from a derivative or progeny thereof, and any combination thereof.

In a related aspect, the invention resides in a stable liquid composition comprising:

(i) one or a plurality of thickening agents, the thickening agents comprising a xanthan gum, inclusive of those described herein, having a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5; and

(ii) a gum, the gum optionally selected from the group consisting of a bean extract, a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof; wherein addition of the composition to an aqueous liquid or aqueous liquid solid mixture foodstuff increases the viscosity of said foodstuff.

The gum of the present aspect is suitably the modified gum hereinbefore described.

Liquid compositions for thickening or increasing the viscosity of a foodstuff are known in the art. By way of example, US2004/ 0197456 (hereinafter “Holahan”) describes a liquid thickener intended for people with swallowing disorders. The invention disclosed in Holahan, however, describes a liquid composition having a thickening agent concentrated to several times its intended usage level. Unlike the controlled-release technology described herein, the liquid thickener of Holahan comprises a thickening agent that already has its viscosity fully expressed therein and, so, which is fully hydrated even before addition to a foodstuff, after which Holahan’s liquid thickener is then simply added at a volume such that the now diluted liquid thickener expresses the desired viscosity in the foodstuff.

In particular embodiments, the stable liquid composition has a water activity of greater than 95%. It would be readily understood, that water activity or a _w is defined as the ratio of the partial vapor pressure of water in a material to the standard state partial vapor pressure of water at the same temperature. Additionally, water generally migrates from areas of high water activity to areas of low water activity. For example, the liquid composition provided herein has a water activity in excess of 95% (e.g., about or in excess of 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% and any range therein), which then typically requires protection from atmospheres or environments with relative humidities of less than 95% so as to prevent the liquid composition from drying out during storage and before delivery or dispensing, such as by a pump dispenser or another sealed delivery system as are known in the art.

The liquid composition of the above aspects may be stored and/or delivered by any means known in the art. In particular embodiments, the liquid composition is stored and/or delivered by a container and pump dispenser arrangement, as are known in the art (see, e.g., PCT/AU2017/050966, which is incorporated by reference herein). In alternative embodiments, the liquid composition is stored and/or delivered by a sachet or the like, such as that provided herein.

Suitably, the liquid composition described herein when added in a desirable amount to an aqueous liquid or aqueous liquid solid mixture foodstuff does not alter particular desirable attributes thereof, such as the original flavour and/or colour of the foodstuff, that may be attractive to the consumer. In this regard, the liquid composition preferably makes little or no flavour and/or colour contribution to said foodstuff when added in a desirable amount thereto. Additionally, it is preferable that the amount of the liquid composition to be added to a foodstuff to achieve a desirable viscosity thereof is as small as possible so as to avoid diluting the flavour and/or colour characteristics of the foodstuff.

With regard to the present invention, the liquid composition described herein is suitably flowable. To this end, the liquid composition of the present invention suitably has a viscosity of less than 4000 cP and more particularly between about 2000 cP to about 4000 cP. Advantageously, a liquid composition of such a viscosity that may be dispensed easily, such as from a pump dispenser or a sachet, as well as being able to be dispersed with little or no agitation (i.e. , a low shear mixing force) when added in a desired amount to an aqueous liquid or aqueous liquid solid mixture foodstuff. Further, the liquid composition of the invention is preferably concentrated and can accommodate a relatively higher percentage of thickening agent without losing the flowable character of the composition. This further enables easy and accurate dispensing of the liquid composition into the foodstuff of choice.

In certain embodiments of the aforementioned aspects, the liquid composition has a viscosity of about 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,

1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000 cP, or any range therein. Suitably, the liquid composition has a viscosity of between about 500 cP to about 1500 cP. More suitably, the liquid composition has a viscosity of between about 750 cP to about 1250 cP.

The viscosity of the liquid composition may be measured by any means known in the art. By way of example, viscosity may be measured using a Bostwick Consistometer, a Brookfield Viscometer, a rheometer or similar device. Suitably, viscosity is measured in absolute centipoise as provided by a rheometer, rather than relative centipoise as measured by a viscometer. It will be appreciated by the skilled artisan that a rheometer measurement represents the best and therefore standard method for determining foodstuff viscosity.

Suitably, the liquid composition described herein increases the viscosity of the aqueous liquid or aqueous liquid solid mixture foodstuff to greater than 95 cP. It is an advantage of the present approach that the inhibition of the expression, by the thickening agent, of its viscosity due to the modified gum is effectively lifted by gentle mixing of the liquid composition into the liquid or liquid solid foodstuff. This allows the thickening agent to quickly express its viscosity, due to the controlled release of the viscosity inhibitory effect of the modified gum on the thickening agent, and therefore aids in its easy and rapid incorporation into the foodstuff. This is an advantage over thickening agents which are substantially fully hydrated prior to being added to a foodstuff, such as that described in Holahan, and can therefore be challenging to incorporate into the foodstuff in a smooth and time efficient manner. Furthermore, the complete expression of viscosity by fully hydrated thickening agents is in itself an obstacle to the easy and rapid development of increased viscosity when it is diluted with a liquid or liquid solid foodstuff.

Therefore, it will be clear that in any of the aforementioned aspects, the thickening agent in the composition is suitably not fully hydrated prior to its addition to the foodstuff.

In certain embodiments, the viscosity of said foodstuff, upon addition of the liquid composition, is increased to at least 95, 100, 110, 120, 130, 140, 150, 175, 200,

250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000 cP, or any range therein.

For the purposes of the present invention, the thickening agent, may be present in an amount from about 3% to about 40% or any range therein such as, but not limited to, about 5% to about 15%, or about 7% to about 12% by weight of the liquid composition. In particular embodiments of the present invention, the thickening agent is present in an amount of about 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%,

18.5%, 19.0%, 19.5%, 20.0%, 20.5%, 21.0%, 21.5%, 22.0%, 22.5%, 23.0%, 23.5%,

24.0%, 24.5%, 25.0%, 25.5%, 26.0%, 26.5%, 27.0%, 27.5%, 28.0%, 28.5%, 29.0%,

29.5%, 30.0%, 30.5%, 31.0%, 31.5%, 32.0%, 32.5%, 33.0%, 33.5%, 34.0%, 34.5%,

35.0%, 35.5%, 36.0%, 36.5%, 37.0%, 37.5%, 38.0%, 38.5%, 39.0%, 39.5%, 40.0% or any range therein, by weight of the liquid composition. In certain embodiments of the present invention, the thickening agent is present in an amount of about 3% to about 20% by weight of the liquid composition.

For the present invention, the modified gum is suitably present in a high enough concentration that does not significantly contribute to the viscosity of the liquid composition. To this end, the modified gum described herein may be present in an amount from about 3% to about 40% or any range therein such as, but not limited to, about 5% to about 20%, or about 7.5% to about 17.5% by weight of the liquid composition.

In particular embodiments of the present invention, the modified gum described herein is present in an amount of about 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%,

6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%,

12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%,

18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20.5%, 21.0%, 21.5%, 22.0%, 22.5%, 23.0%,

23.5%, 24.0%, 24.5%, 25.0%, 25.5%, 26.0%, 26.5%, 27.0%, 27.5%, 28.0%, 28.5%,

29.0%, 29.5%, 30.0%, 30.5%, 31.0%, 31.5%, 32.0%, 32.5%, 33.0%, 33.5%, 34.0%,

34.5%, 35.0%, 35.5%, 36.0%, 36.5%, 37.0%, 37.5%, 38.0%, 38.5%, 39.0%, 39.5%, 40.0% or any range therein, by weight of the liquid composition. In certain embodiments of the present invention, the modified gum described herein is present in an amount of about 3% to about 20% by weight of the liquid composition.

Suitably, the modified gum is included in an amount such that the stable liquid composition has a lower viscosity than that of the liquid composition were it to comprise the thickening agent only with water or another suitable aqueous solution. More suitably, the modified gum decreases the viscosity of the stable liquid composition to at least a third of that of the liquid composition were it to comprise the thickening agent only with water or another suitable aqueous solution. In particular embodiments, the modified gum decreases the viscosity of the stable liquid composition to at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,

16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or any range therein, of that of the liquid composition were it to comprise the thickening agent only with water or another suitable aqueous solution.

For the purposes of the present invention, the stable liquid composition suitably comprises potassium chloride at a concentration of about 0.01 mol/L to about 0.5 mol/L or any range therein such as, but not limited to, about 0.02 mol/L to about 0.25 mol/L, or about 0.05 mol/L to about 0.10 mol/L. In particular embodiments of the present invention, the thickening agent is present at a concentration of about 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 mol/L or any range therein, in the liquid composition. In certain embodiments of the present invention, the potassium chloride is present at a concentration of about 0.02 mol/L to about 0.1 mol/L in the liquid composition.

Suitably, the stable liquid composition described herein has a Brix value of about 10° Bx to about 40° Bx (e.g., 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15,

15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21 , 21.5, 22, 22.5, 23, 23.5, 24,

24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31 , 31.5, 32, 32.5, 33,

33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40° Bx or any range therein). In this regard, the Brix value or content refers to the dissolved solid content of an aqueous solution. The Brix value may be measured by any means known in the art, such as refractometer, hydrometer or density meter.

Suitably, the composition referred to herein is stable for at least six months and up to at least two years at room temperature. In this regard, the inventors have shown that the present liquid composition including the modified gum demonstrates little or no separation between its component materials (e.g., the modified gum and the thickening agent) after storage at room temperature for 6 months or more. This is in contrast to those liquid thickening agents known in the art. By way of example, US Patent 6,455,090 (hereinafter “Uzahashi”) describes methods for producing a liquid thickening formulation, which can thicken when added to a liquid and is initially inhibited from forming viscous solutions or gels. The inventors claim that the invention can be added suitably to a liquid or semi-liquid foodstuff for a patient who has mastication and deglutition difficulties.

Nonetheless, the invention disclosed in Uzahashi is limited in that the thickening agent described therein exhibits neither microbial nor physically stability, but rather rapidly separates to create layers. Additionally, the thickening agent of Uzahashi fails to consistently and uniformly thicken liquid foods when added thereto. As such, the liquid thickener of Uzahashi has no practical utility in the management of swallowing disorders (dysphagia) so as to prevent or limit common co-morbidities of the condition. This lack of utility is two-fold. Firstly, the lack of physical stability and resultant separation of the solvent and gelling agents prohibits accurate dosing of Uzahashi’s liquid thickener. As such the invention as disclosed cannot consistently guarantee to meet the required levels with regard to predetermined viscosity of the resultant thickened food. Secondly, patients such as those described herein are typically vulnerable populations. Indeed, the liquid thickener composition of Uzahashi is not microbiologically stable and thus should not be administered clinically to the intended population as described. Conversely, the liquid composition including the modified gum described herein successfully overcomes this limitation of the prior art by not separating to create layers and thus consistently imparts an accurate pre-determ ined viscosity to an aqueous liquid or aqueous liquid solid mixture foodstuff when added thereto (see, e.g., Table 3). Because the composition of the present invention is stable, without significant degradation in the performance of the thickening agent, the viscosity remains constant for a commercially reasonable period of time. Accordingly, the formulation can be provided as a packaged product perse, such as in a metered pump dispenser or in a sachet, to the end user. To this end, the end user can reliably calculate the amount of the liquid composition of the invention to add to a food or beverage to achieve a desired end viscosity thereof. The liquid composition of the invention is then easily dispensed and easily mixed into the foodstuff to give the desired end product.

As described earlier, the ability to package and use the liquid composition in this way is a result of the combined presence of the thickening agent and modified gum which inhibits the expression of the viscosity of the thickening agent until released through the application of low shear mixing and provides distinct benefits in use over traditional sachets of powdered or gel-like thickener which are notoriously difficult to measure out accurately, when the exact pack size is not appropriate, and to incorporate into liquid foodstuffs.

Stability of the liquid composition of the invention over time may be indicated by the retention of colour (if any), flavour (if any), separation (if any), microbiological spoilage (if any), viscosity and/or clarity of the liquid composition. Additionally or alternatively, stability of the liquid composition may be determined by the ability of the composition to impart viscosity consistently and repeatably to a predetermined level when added to a foodstuff. The stability of the liquid composition can be determined by using any of the techniques available to a person skilled in art of food science, including microbiological testing to measure the extent and rate of microbiological spoilage; visual inspection for physical changes such as separation and/or sedimentation; sensory evaluation to determine colour, flavour and/or clarity changes; and viscosity measurement using a Bostwick Consistometer, Brookfield Viscometer, a rheometer or similar device.

With respect to stability, the liquid composition of the invention may further comprise a food-grade preservative and/or stabilizer, as are well known in the art.

Suitable food grade preservatives and stabilizers include, but are not limited to, gellan gum, vitamin E, potassium sorbate, sodium benzoate, sodiummetabisulphite, methyl paraben, EDTA, sulphur dioxide, nisin and propionic acid. In various embodiments, the food-grade preservative/stabilizer is or comprises gellan gum. The amount of preservative in the liquid composition may range from about 0.001 to about 0.1 percent by weight of the total weight of the liquid composition.

Again, in regards to stability, the liquid composition described herein suitably is of a pH between about 3.0 and about 7.5 (e.g., 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5 and any range therein). In particular embodiments, the pH of the liquid composition is between about 4 and 4.4. To this end, the acidic pH of the liquid composition may be achieved by any means known in the art, such as those hereinbefore described.

Suitably, the liquid composition described herein is added to an aqueous liquid or aqueous liquid solid mixture foodstuff for feeding to a subject suffering from a mastication and/or deglutition disease, disorder or condition. Suitably, the mastication and/or deglutition disease, disorder or condition is or comprises dysphagia. As such, it is preferable for this use that the liquid composition is separated into appropriate individual portions, such as sachets, or be pump dispensable.

It will be readily understood that dysphagia is a condition where the process of swallowing is impaired. During eating, this can lead to the entry of liquid or solid food into the trachea and subsequently the lungs of the sufferer potentially leading to aspiration pneumonia. Dysphagia can occur at any age, but is most common in the elderly, especially if they have suffered a stroke or have dementia. One management strategy for sufferers of dysphagia is to consume foods that are texture modified (i.e. , thickened foods and beverages) that slow the swallowing reflex and allow the windpipe time to close before the food passes, thereby preventing aspiration of food.

In yet another aspect, the invention provides a method of producing a stable liquid composition, including the steps of:

(i) providing a modified gum described herein;

(ii) adding one or a plurality of thickening agents to the modified gum; and

(iii) mixing the mixture of step (ii) to thereby produce the stable liquid composition.

Accordingly, in some embodiments, the present method may include the step of preparing the modified gum, such as by way of those steps hereinbefore described. In a related aspect, the invention resides in a method of producing a stable liquid composition, including the steps of:

(i) providing one or a plurality of thickening agents, the thickening agents comprising a xanthan gum having a pyruvic acid content of at least 5.0% (w/w) and a pyruvic acid to acetic acid w/w ratio of at least 0.5.; and

(ii) adding a gum, the gum optionally selected from the group consisting of a bean extract, a Larix occidentalis polysaccharide extract, a Larix laricina polysaccharide extract, an Acacia tree polysaccharide extract, a Larix decidua polysaccharide extract, a Larix sibirica polysaccharide extract and any combination thereof to the thickening agents; and

(iii) mixing the mixture of step (ii) to thereby produce the stable liquid composition.

Suitably, the stable liquid composition is that hereinbefore described.

Manufacture of the stable liquid composition of the invention can include the step of heating the gum or modified gum and/or the one or plurality of thickening agents when present, for example, in a suitable liquid carrier, such as an aqueous carrier. The heated composition can then be hot-fill packaged, or cooled prior to packaging.

The present method may include the step of preparing an aqueous solution or suspension of the modified gum. In this regard, the aqueous solution may have a dry mass content of the modified gum from about 0.1 to about 60 wt%, based on the total amount of the aqueous solution of suspension.

Similarly, the present method may include the step of preparing an aqueous solution or suspension of the thickening agent. In this regard, the aqueous solution may have a dry mass content of the thickening agent from about 0.1 to about 60 wt%, based on the total amount of the aqueous solution of suspension.

In one embodiment, the method of the present aspect further includes the step of adjusting the pH of the stable liquid composition to a pH of about 3.5 to about 4.5.

In some embodiments, the current method further includes the step of adding potassium chloride to a concentration of about 0.01 mol/L to about 0.5 mol/L.

In other embodiments, the method of the present aspect further includes the step of adjusting a Brix value of the stable liquid composition to about 10° Bx to about 40° Bx. By way of example, the present step may include adding a sugar or other solute to the stable liquid composition at a concentration sufficient to cause the stable liquid composition to have a Brix value of about 10° Bx to about 40° Bx.

The method of the current aspect may optionally include the step of adding one or more excipients or additives to the stable liquid composition, such as colours, flavours, protein (animal and plant), dietary fibres, vitamins and minerals, humectants, for example glycerol and sorbitol, fats and oils, emulsifiers, acidity regulators, antioxidants, low calorie bulking agents, firming agents, flavour enhancers, foaming agents, gelling agents, preservatives, sequestrants and stabilisers.

In yet a further aspect, the invention provides a method for increasing the viscosity of an aqueous liquid or aqueous liquid solid mixture foodstuff, the method including the steps of:

(a) adding to the foodstuff a stable liquid composition described herein; and

(b) mixing the foodstuff and the composition so as to promote increasing the viscosity of said foodstuff by the composition.

In particular embodiments, a level turbidity of the foodstuff to which the stable liquid composition of the present aspect has been added is reduced by at least 5% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% etc and any range therein) when compared to that of a liquid that has been thickened by an equivalent liquid composition that includes a gum and/or thickening agent that has not been modified in the manner described herein. Relevant levels of turbidity or clarity may be assessed by standard means known in the art, such as a nephelometer or turbidimeter and including those described herein.

Suitably, the method further comprises the step of applying low-shear mixing to the foodstuff and the composition so as to promote increasing the viscosity of said foodstuff by the composition.

As generally used herein, the term "low shear mixing" refers to non-turbulent or minimally turbulent mixing, such as gentle mixing or stirring with a spoon or the like. It would be understood that low-shear mixing may be defined in terms of shear rates and typically is a function of a number of variables, such as mixing vessel configuration and mixing device speed. It will be appreciated that the low-shear mixing is suitably of a value that is sufficient to promote the physical removal of the gum or modified gum from its inhibitory interaction site on the one or plurality of thickening agents, so as to allow said thickening agents to exert their desired effect of increasing the relevant liquid or semi-liquid’s foodstuffs viscosity. Accordingly, in particular embodiments the low- shear mixing comprises stirring at a speed of from about 10 rpm to about 150 rpm (e.g., about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 rpm or any range therein).

Suitably, the low-shear mixing is applied for about 60 seconds or less to achieve a maximal or near-maximal increase in viscosity of the foodstuff. Suitably, the low- shear mixing is applied for about 10 to about 40 seconds (e.g., about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 seconds or any range therein) to achieve a maximal or near maximal viscosity of the foodstuff.

In certain embodiments, the viscosity of the foodstuff is suitably increased to greater than 95 cP.

In referring to the above aspect, the foodstuff of increased viscosity is suitably for feeding a subject suffering from a mastication and/or deglutition disease, disorder or condition. Suitably, the mastication and/or deglutition disease, disorder or condition is or comprises dysphagia.

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

In order that the invention may be more readily understood and put into practice, one or more preferred embodiments thereof will now be described, by way of example only.

Example 1 - Initial clarity assessments The objective of the present example was to investigate the mechanism behind the undesirable haziness or cloudiness that can be observed for viscosity-inhibited liquid thickeners when added to a clear liquid, such as water. In particular, we have analysed the interaction that seemingly occurs between the polysaccharide- or gum- based ingredient (e.g., gum arabic) and the thickening agent (e.g., xanthan gum) inhibited thereby.

For the present embodiment, a viscosity-inhibited liquid thickener product containing appropriate amounts of inhibitory gum as the polysaccharide-based ingredient (that inhibits the thickening agent in increasing viscosity) and xanthan gum as the thickening agent has been investigated. In this regard, Figure 1 demonstrates that the inhibitory gum somehow interacts with the xanthan gum as it unfurls from its native state during partial hydration causing a colloidal haze (see samples G & H). Xanthan gum as it partially unfurls from its native state during processing does not cause a colloidal haze as neither the inhibitory gum rich solution, the fully hydrated xanthan solution or equivalent admixtures of the inhibitory gum rich solution and fully hydrated xanthan mixed at equivalent concentrations to a 5% solution of the viscosity inhibited concentration exhibit colloidal haze similar to dilutions of the viscosity inhibited liquid thickener (Figure 1 ; Table 1 ).

Table 1 % Transmittance (650 nm) and Absorbance of a 5% w/v liquid thickener solution in water compared to a fully hydrated xanthan solution, a inhibitory gum rich solution, equivalent mixtures of the inhibitory gum solution and fully hydrated xanthan solution (i.e. , undiluted liquid thickener) prepared either hot or cold. ‘Additive effect of individual components Initial microscopic evaluation using 40X magnification with light microscope of 5% w/v aqueous solutions of the viscosity-inhibited liquid thickener suggest that molecular interactions occur between the polysaccharide-based ingredient (i.e. , the inhibitory gum) and the thickening agent (i.e., the xanthan gum), with thin fibrous insoluble particles visible in these solutions (i.e., samples G and H of Table 1 and Figure 1 ) (Figure 2), which were not visible in either of the concentrated solutions containing xanthan gum and/or inhibitory gum.

Example 2 - Effect of pH on clarity

The present Example investigated whether changes in pH of the concentrated liquid thickener (i.e., prior to dilution) could improve the clarity of clear liquids thickened thereby. In this regard, the loss of clarity could be the result of insoluble complexes forming between anionic branch chains of the liquid thickener (e.g., acetate or pyruvate or both) and glycoprotein branches of the polysaccharide-based ingredient during dilution of the liquid thickener. As such, an electrical charge equivalence pH (EEP) may be evident at the isoelectric point of the combined charged molecules, with a greater clarity evident at the theoretical EEP.

Batches of xanthan gum in inhibitory gum were made at various pHs between 3-10 and the diluted product assessed for clarity/haziness objectively using an absorbance reading on a spectrophotometer after preparation.

A minimum absorbance was obvious on the pH vs absorbance plot between a pH of 4 and 4.1 (Figure 3). Thus, a liquid thickener concentrate formulated with a pH of 4.0-4.1 results in clearer beverages with improved clarity, although as shown in Figure 3 further increases in pH only produce minor reductions in clarity.

Example 3 - Effect of Ionic Adjuncts on Clarity

The effective life of xanthan solutions can be extended through addition of 1.5% sodium erythrobate or 1 .5% sodium ascorbate. This is attributed to prevention of cross linking of xanthan charged side branches with iron. It was hypothesised that a similar effect might be achieved in preventing the liquid thickener cross linking with the glycoprotein or arabinogalactan-protein (AGP) on the polysaccharide-based ingredient and result in reduced production of insoluble complexes and hence increased clarity and reduced turbidity.

Further to the above, solutes or salts such as urea and sodium chloride at concentrations between 0.1 mol/L and 0.5 mol/L may reduce turbidity due to inhibition of the formation of complex coacervates in model systems containing xanthan gum and whey protein. As such, it was tested whether the loss of clarity (turbidity) in water thickened with a viscosity-inhibited liquid thickener results from the formation of complex coacervates between glycoprotein of the polysaccharide-based ingredient and anionic side chains of the thickening agents. To this end, it was assessed whether the inclusion of solutes (i.e. , KCI) in the viscosity-inhibited liquid thickener in a concentration range between 0.1 and 0.5 mol/L would result in thickened water with improved clarity and less turbidity.

It was hypothesised that an inhibition of turbidity as a result of addition of KCI would be greater due to the comparatively larger molecular size of the molecule and thus increased steric hindrance of cationic and anionic side chains interacting.

Batches of concentrated xanthan gum and concentrated inhibitory gum were made with the following solvents:

• Tap water

• Sodium chloride solutions in the range of 0.01 M to 0.5M

• Potassium chloride solutions in the range of 0.01 M to 0.5M

• Sodium erythrobate 1 .5%

• Sodium ascorbate 1 .5%

• Urea solutions

These batches of the viscosity-inhibited liquid thickener were then diluted in water (5% w/v) to produce IDDSI Level 2 Mildly Thick consistencies and assessed for clarity/haziness visually, and objectively using turbidity absorbance readings on a spectrophotometer after preparation.

Turbidity and absorbance at 395nm were not significantly affected by the addition of solutes with the exception of KCL which showed distinct improvements in minimum absorbance and turbidity. These results in keeping with the hypothesis that the larger molecular weight potassium molecule could sterically hinder formation of insoluble complexes and result in improved clarity.

Example 4 - Optimisation of KCI addition

As the viscosity inhibited liquid thickener is intended for vulnerable populations an attempt was made to optimise clarity using concentrations of solute that would not represent a potentially unnecessarily high renal solute load. Concentrations between 0.02 and 0.1 mol/L of potassium chloride were added to the viscosity inhibited thickener during manufacture prior to addition of xanthan gum.

Spectrophotometer results indicate (Figure 4) a peak transmittance at 0.09mol/L. Nonetheless, all concentrations of KCI tested produced an improvement in clarity as demonstrated in Figure 5.

Example 5 - Optimisation of solids

It was demonstrated that controlling for total soluble solids in the viscosity- inhibited concentrate showed similar effects on peak absorbance as the addition of 0.25 mol/L equivalent amounts of KCI. Improved clarity (as determined by lower turbidity and lower peak absorbance at 395nm) was observed when adjusting the level of total soluble solids in the viscosity-inhibited liquid thickener to between about 24°Brix and 26°Brix, such as by varying the amount of gum or thickening agent therein or by diluting standard concentrated solutions for producing the liquid thickener.

Example 6 - Other acidulants and removal of Potassium Sorbate

Acidulants such as sodium acid sulphate and phosphoric acid rapidly decrease solution pH. In clear beverage applications containing whey proteins the use of such acidulants is preferred as whey proteins become fully soluble (as evident of solution clarity) when the solution pH is below 3 and most preferable below pH 2.7-2.8. Acidulants that rapidly move the whey protein past its isoelectric point of 4.3 result in minimal formation of insolubles when the protein is at its EEP and thus produce less turbid solutions. It was hypothesised that the empirical observations on model xanthan systems could follow a similar chemistry. It is possible that use of these will not have a similar effect on turbidity in the viscosity inhibited system as the turbidity described is not consistent with the complex coacervation theory (different chemistry).

As hypothesised no evidence of improved clarity was observed for either acidulant (note all trial variants targeted optimal pH and total solids as determined in above Examples).

Example 7 - Use of retarded xanthan

Industrial applications for xanthan such as the paint industry employ the use of retarded xanthan's which control the release of the viscosity by inhibiting hydration until such times as it is ‘triggered’ either by a change in pH or by 'melting' away the encapsulant with increased temperatures. It was considered that the use of a similar system may inhibit the formation of the insoluble substructures observed in diluted solutions of the viscosity inhibited liquid as the opportunity for formation of these will be minimised as the of a retarded xanthan should significantly reduce the time required to produce the viscosity inhibited system and thus significantly reduce total interaction time between haze active particles. This avenue of development was discontinued as it was found that such xanthans were incompatible with a viscosity-inhibited liquid thickener system and unable to be ‘released’ and exert their viscosity at the end of processing.

Example 8 - Effect of different sources of the polysaccharide-based ingredient with varying protein levels

There can be differing protein contents (Table 2) amongst different species of gum arabic sources approved for use in food. Thus, if the insoluble haze particles are protein complexes with anionic side chains on the xanthan gum, it is possible that use of a species of Acacia gum with lower expected protein content in the viscosity- inhibited liquid thickener system would result in the formation of less insoluble protein complexes and hence improved clarity.

Table 2 Levels of protein reported in gum Arabic.

Interestingly there was no evidence of improved clarity when using reduced protein species of Gum Acacia even when accounting for twice the turbidity in the native inhibitory gum solution (289 FTU) as compared to commercial supply (140 FTU), with turbidity measured in 5% diluted concentrate solutions being significantly higher (Table 3) for dilutions of the liquid thickener concentrate made with lower protein gum arabic sources.

Table 3 Turbidity measure in 5% diluted concentrate solution control and trial variants at optimal pH (4.1) and total solids (24° Brix).

Subsequently a ‘clearer’ source of A Senegal with significant amounts of protein removed during gum processing and drying was obtained and trailed and compared to material from the same supplier with a native Gum protein content. Concentrated solutions of the inhibitory gum were produced and subsequently diluted to an equivalent concentration as present in a 5% dilution of the viscosity inhibited liquid thickener from both the protein reduced Gum Arabic and standard commercial Gum Arabic. The two concentrate solutions showed substantially different absorbance and transmittance at 650 nm (Table 4) with the thickener concentrate having the lower protein gum arabic showing lower absorbance and % transmittance than the non- protein modified control thickener. Interestingly this trial product appeared visually to be clearer (Figure 7) Table 4 Absorbance and % Transmittance measured in 4.32% process intermediate solution produced from control and trial variants of inhibitory gum Surprisingly, there was no evidence if improved clarity when using reduced protein gum arabic as compared to standard commercial supply material with absorbance and % transmittance measure in 5% diluted concentrate solution substantially equivalent (Table 5; Figure 8).

Table 5 Absorbance and % Transmittance measured in 5% diluted concentrate solution control and trial variants at optimal pH (4.1) and total solids (24° Brix).

Thus, we conclude that turbidity/haze in application is not related to absolute concentration of protein in the system and can't be improved by simply reducing the threshold amount of protein in the system other factors clearly impact on clarity. Note all trial variants targeted optimal pH and total solids and clearest grade of xanthan as determined in the above Examples.

Example 9 - Use of beverage clarifiers

If the presently observed haze/lack of clarity is the result of protein-complex formation between a protein rich fraction of the polysaccharide-based ingredient and charged molecules on branch chains of the thickening agent then it may be possible to apply methods of stabilisation used in clarifying beverages such as beer (adsorption, precipitation and enzymatic hydrolysation) to overcome colloidal haze (e.g., polyphenol complexes with protein) in brewing by removing either proteins of molecular weight > 40000D, polyphenols or both may also have application to overcome haze/loss of clarity in viscosity-inhibited liquid thickener systems.

Adsorption trials:

Aluminium silicate (bentonite) treatment was applied to a concentrated inhibitory gum solution adjusted to pH 4.9 with NaOH. The solution was treated by heating to 80°C and bentonite added at rate of 3g/100HL. The solution was whirlpooled for 2 hours. Brix were adjusted 16° to account for water vapour lost during treatment so as the target optimal 24°Brix was achieved in the viscosity inhibited liquid concentrate.

No evidence if improved clarity (Table 6) was observed. Thus, we conclude that haze development in viscosity inhibited system is not the same mechanism as polyphenol cross linking underpinning haze in beer.

Table 6 Turbidity measure in 5% diluted concentrate solution control and trial variants at optimal pH (4.1) and total solids (24° Brix).

Precipitation trials Carrageenan and tannic acid are used on beer to reduce or eliminate colloidal protein haze. Thus, carrageenan and tannic acid may preferentially bind to the protein fractions of Gum Acacia and thus precipitate out of solution on application of whirlpool. The subsequent protein reduced solution may therefore produce less insoluble protein complexes and thus have less turbidity and greater clarity. Tannic acid has many hydroxyl groups that attract nucleophilic SH- and NH- groups of haze active proteins in applications such as beer. Once bound these can be precipitated out of solution using a vortex. Carrageenan works on same principle.

Thus, Tannic acid (Brewtan) or Carrageenan (Whirl Floe) treatments either in isolation or in combination were applied to a concentrated inhibitory gum solution adjusted to pH 2 or 4.5 with NaOH and heated to 70-80°C. Whirl floe was added at rate of 1.2g/6L and Brewtan added at rate of 0.35g/6L and all treatments were held under whirlpool for 2 hours.

Table 7 Turbidity measure in 5% diluted concentrate solution control and trial variants at optimal pH (4.1) and total solids (24° Brix).

Combined application of Whirlfloc and Brewtan in an inhibitory gum process intermediate adjusted to pH 4.5 resulted in the lowest turbidity and absorbance, however, this was only a marginal improvement in clarity as compared to un-treated control (Table 7).

Thus, as removal of haze-active proteins with silica gel absorbing proteins and tannin acid titration did not eliminate or significantly reduce the haze in the viscosity- inhibited liquid thickener upon dilution, we conclude the ‘haze’ in viscosity-inhibited liquid thickener systems differs from that responsible for haze in beer, wine and beverages. Enzyme Hydrolysis trials

Non-biological turbidity is well known in modern stabilization practices in the beverage industry (beer, wine and fruit juice) and referred to as “colloidal haze". It is caused by low molecular weight polyphenols cross-linking with proteins by weak molecular interactions or protein-protein interactions. The majority (82%) of proteins implicated in this non biological turbidity in brewing are known to have a molecular weight of 13 k Daltons or less (Table 8).

Table 8. Protein fractions in beer and their tendency to form turbidity. Source: Mussche, De Pauw: J Inst Brewing 1999.

In brewing, Papain, a broad acting protease produced from the latex of Carica papaya, can be added at a rate of 2 to 6 ml/hl (0.02-0.06%) to rough or bright beer to hydrolyse both haze active and non-haze active proteins thus preventing interactions with other proteins and organic components in the beer (polyphenols and tannins). Degrading these proteins prevents the formation of large complex aggregates with polyphenols which become a visible haze in beer.

The use of prolyl-specific endoproteases for the prevention or reduction of haze in beverages has been described. US 8119171B2 describes a proteolytic method to reduce haze in a beverages (beer, beer wort, wine and fruit juice). Specifically, it teaches a method of adding a proline-specific endoprotease protein-containing preparation to the beverage, wherein the proline-specific endoprotease hydrolyses a protein or peptide at places where the protein or peptide contains a prolyl residue to proteolytically reduce haze in the beverage. CN 101 294 53 describes a proline-specific endopeptidase, which can specifically degrade (proline-rich) proteins or small proteins which are the haze- sensitive proteins in the brewing process. This patent relates to a Aspergillus niger proline-specific endoprotease which can specifically cut protein and proline residues at a terminal carbon of a peptide chain which eliminates cold haze substance in beer. Specifically, it claims that haze protein bands with molecular weight of 8 to 14 kDa disappeared completely after SDS-polyacrylamide gel electrophoresis detection of beer protein treated with PEP, “which shows that the haze activity protein rich in proline residues is hydrolysed completely by PEP”.

The amino acid profile of gum arabic from Acacia Senegal is predominantly hydroxyproline and serine (Table 9). Further, we have shown that the starting inhibitory gum material (Figure 9, lane 8), when subjected to SDS PAGE according to Laemmli only has one strong protein band at 20 kDa and two faint bands at 40 and 60 kDa and no bands with molecular weight of 8 to 14 kDa determined to be responsible for beer protein haze according to CN 101 294 53. Furthermore, polyphenols are synthesized by plants. The production of polyphenols in an industrial bio fermentation process, such as Xanthomonas to produce xanthan gum, would require a genetically modified Xanthomonas spp with functional integration of biosynthetic plant polyphenol pathways into the microorganisms to express polyphenols. Thus, the underlying mechanism explaining the colloidal haze in the viscosity inhibited liquid thickening agent cannot be explained as above for beverage applications such as beer.

Table 9 Amino acid composition of Acacia Senegal and Acacia Seyal gums (residues/1000 molecules). Source: P.A. Williams, G.O. Phillips, in Handbook of Hydrocolloids (Second Edition), 2009

Given the incompatibility of the substrate materials in viscosity-inhibited liquid thickener systems with well described mechanisms in the public domain for colloidal haze in beverages such as beer, wine and juice, it is not surprising that the use of prolyl-specific endoproteases alone or in combination with other enzymes for reduction of ‘haze’ in dilutions of viscosity inhibited mixed hydrocolloid sols has never, to our knowledge, been described or suggested.

Adding 0.025% papain to a concentrated inhibitory gum solution (0.38% protein) adjusted to pH 6.5 and treated for 3, 5 or 7 hours at (80C) resulted in moderate improvements in clarity as compared to an untreated control (Table 11; Figure 10). These results are surprising, as the relatively minor Arabinogalactan (AG) fraction of Gum Arabic is well known to be resistant to papain due to the inaccessibility of the peptide backbone and the remarkable stability of this fraction in the alkaline conditions favouring papain activity (pH 6-7). However, the major AGP protein component, is known to be cleaved using enzymes in alkaline conditions (Renard et al 2014). Thus, a significant reduction in visible protein colloidal haze was expected for this variant, whilst only a moderate improvement was observed. Table 10 - % Transmittance (650 nm) and IDDSI viscosity of 5% liquid thickener solution in water (IDDSI L2).

Further to the above, adding 0.06% proline specific endopeptidase to a concentrated inhibitory gum solution (0.38% protein) adjusted to pH 5.2 and treated for up to 72 hours at ambient temperature results in moderate improvements in clarity as compared to an untreated control (Table 11) and comparable to improvements seen with papain treatment (Table 10; Figure 11). Table 11 % Transmittance and absorbance (650 nm) of 5% liquid thickener solution in water (IDDSI L2) prepared from process intermediate treated with proline specific endopeptidase. Sequential Enzymic treatments

Following on from the above, enzymatic treatments of papain and proline specific endopeptidase were applied sequentially to see if the differing targeted activity subjected to different treatment temperatures and concentrations with and without optimised concentrations of ionic adjuncts could provide at least an additive effect in preventing the formation of haze in a viscosity-inhibited liquid thickener system whilst also retaining viscosity. Sequential enzymic treatments enzyme concentration dependence

Adding 0.06% proline specific endopeptidase to a concentrated inhibitory gum solution (0.38% protein) adjusted to pH 5.2 and treated up to 72 hours at ambient temperature followed by treatment with papain at varying times (1.5-3 hours) and concentrations (0.026-0.051%) at pH 6.5 and 80°C resulted in improved clarity as compared to an untreated control (Table 12).

Table 12 Percentage (%) Transmittance (650 nm) and IDDSI viscosity of 5% liquid thickener solution in water (IDDSI L2) prepared from process intermediate sequentially treated with proline specific endopeptidase and papain.

Sequential Enzymic treatments treatment time dependence

Adding 0.06% proline specific endopeptidase to a concentrated inhibitory gum solution (0.38% protein) adjusted to pH 5.2 and treated for up to 24 hours at ambient temperature followed by treatment with 0.026% papain treatment at 80°C for 3 hours produced significant improvements in transmittance across the entire treatment period tested. There was, however, quite a defined peak transmittance at 12-13 hours of treatment (Figure 11). The reason for this is unclear, however, it is hypothesised that peak transmittance correlates with a hydrolysed fraction that has increased inaccessibility of the peptide backbone to the anionic side chain of the xanthan molecule. Sequential Enzymic treatments time dependence with 0.045mol/L KCI and 10 times concentration of Papain.

Adding 0.06% proline specific endopeptidase to a concentrated inhibitory gum solution (0.38% protein) containing 0.045mol/L KCI and adjusted to pH 5.2 and treated for between and 12 and 40 hours at ambient temperature followed by treatment with 0.26% papain treatment at 80°C for 3 hours produced significant improvements in transmittance across the entire treatment period tested. There was, however, a defined peak transmittance at 15-16 hours treatment (Figure 12). The reason for this is unclear, however, again it hypothesised that peak transmittance correlates with a hydrolysed fraction that has increased inaccessibility of the peptide backbone to the anionic side chain of the xanthan molecule and accessibility is further hindered by the large potassium ion in solution.

Sequential Enzyme treatments in reverse order of addition (i.e., papain before proline specific endopeptidase)

Adding 0.26% papain treatment to a concentrated inhibitory gum solution (0.38% protein) containing -.045mol/L KCI and adjusted to pH 7.5 at 80°C for 3 to 7 hours followed by 0.06% proline specific endopeptidase treatment and treated for up to 24 hours at 60°C resulted in a defined peak transmittance at 19 hours treatment (Table 13 below). The reason for this is unclear, however, again it hypothesised that peak transmittance correlates with a hydrolysed fraction that has increased inaccessibility of the peptide backbone to the anionic side chain of the xanthan molecule and accessibility is further hindered by the large potassium ion in solution.

Notwithstanding the above, all of the treatment variables tested produced improvements in clarity for a viscosity-inhibited liquid thickener produced therefrom, as compared to that produced from untreated control inhibitory gums.

Results

Table 13 - Sequential treatment with Papain and then Clarex trials Sequential Enzymic treatments, with ionic adjuncts and use of clearer inhibitory gum with altered protein content.

The “clearer” source of Acacia Senegal with significant amounts of protein removed during gum processing and drying was obtained and trialled as follows: Adding 0.06% proline specific endopeptidase to the concentrated inhibitory gum solution (reduced % protein) containing 0.045mol/L KCI and adjusted to pH 5.2 and treated for between and 12 and 40 hours at ambient temperature followed by treatment with 0.26% papain treatment at 80°C for 3 hours. While clarity was significantly improved with these trial variants (Table 14;

Figure 13), a colloidal haze was still visible when 5% solutions of the viscosity inhibited liquid thickener were prepared with hot and cold water (Figure 14).

Table 14 % Transmittance (650 nm) and IDDSI viscosity of 5% liquid thickener solution in water (IDDSI L2) prepared from concentrated inhibitory gum (protein reduced at production) process intermediate treated sequentially with proline specific endopeptidase and papain.

Additional Enzyme Trials - Additional proteases and carbohydrase

A number of additional enzymes were added at 0.05% singly or in combination (as described in Table 15, 16 and 17 below) to a concentrated inhibitory gum solution (0.38% protein) containing 0.045mol/L KCI and adjusted to optimal pH (as described below) and treated for between 4 and 20 hours at optimal temperatures described. After required run time, pH adjusted to 3.9 and viscosity inhibited concentrate made with desired xanthan gum. As demonstrated in Table 16, each of the combination treatments for the enzymes tested demonstrated improvements in clarity when compared to untreated controls (i.e., untreated inhibitory gum). The serine specific endopeptidase Alcalase (0.05%) treated in combination with papain (0.26%) for 20 hours at pH 8 and 80° resulted in a defined peak transmittance of 80.1 at 650 nm (Table 16). Treating with the proline specific Clarex for 16HR (at pH 5.2 and 60°C) followed by Alcalase treatment for 4HR (at H 8 and 70°C) also resulted in a peak transmittance of 78.8 at 650 nm.

Interestingly, Clarex and Alcalase enzymes hydrolyse the two most relatively abundant amino acids in the inhibitory gum protein fraction but result in lower peak transmittance when used in combination as compared when used individually or in combination with papain, a cysteine protease and the second least abundant amino acid in the inhibitory gum protein fraction. Further, use of a carbohydrase (viscoenzyme) for 20 hours under optimal conditions resulted in significantly improved clarity suggesting a complex mechanism underlying turbidity in this application.

Table 15 - Enzymes

Results

Table 16 - Individual enzyme trials Table 17 - Further enzyme combination trials

Sequential Enzymic treatments, with ionic adjuncts and use of novel xanthan gums with altered charge density

Xanthan gum produced commercially by Xanthomonas campestris is a right- handed, five-fold helix conformation where the charged side chains are aligned with the backbone. In solution, the side chains wrap around the backbone. US 9,380,803 describes the production of Xanthomonas sp. with defective biosynthetic pathways that produced xanthan gums that vary in the content and position of the acetate and pyruvate groups and have high viscosity and excellent synergy in admixtures with galactomannans and glucomannans.

We tested whether such a xanthan gum with altered content and position of the acetate and pyruvate groups could exhibit a ‘Detergent effect’ in the described application. That is, the combination of hydrolysed inhibitory gum and the altered charge density of the novel xanthan alter the solution charge density and in doing so optimise the solubility of components (possibly the glycoproteins) that might be sitting in the gas/liquid interphase. An alternate possibility is that the variations in the content and position of the acetate and pyruvate groups on partial unfurling allow an interaction with the enzyme-hydrolysed inhibitory gum so that the polysaccharide strands are in a linear or non-clustered alignment facilitating more transmittance of light.

Five percent solutions of the enzyme pre-treated viscosity inhibited liquid thickener produced using a xanthan gum having a pyruvic acid content > 5% and a pyruvic acid to acetic acid ratio of > 0.5 show improved clarity as determined by the absence of insoluble particles as compared to both control and equivalent solutions produced from standard high clarity xanthan gums suspended in enzyme and non- enzyme treated inhibitory gum solutions (Figure 15). Importantly the use of this modified xanthan gum showed no evidence of the insoluble haze particles which remain even after extended heating, cooling and centrifugation of the five percent solution of the enzyme pre-treated viscosity inhibited liquid thickener produced using commercial xanthan gum containing 3-4% acetate and 4-5% pyruvate (Figure 16). Example 11 - Protein Analysis of Modified Gum

The aim of this Example was to firstly characterise the modified gum at each step of the process to demonstrate the physical changes occurring in the material during production/processing, and secondly to test the proposed hypotheses with the aim of explaining the mechanism behind the clarity observed with this modified hydrocolloid.

Sample information

Seventeen modified gum samples were analysed (see Table 18).

Table 18 - List of gum samples for viscosity and DH analysis Viscosity analysis

Viscosity measurements were carried out in a Brookfield DV3T Rheometer with a small sample adaptor. The samples were made up to a 5% w/w concentration in R.O. water. Each sample was then ultra-sonicated for approximately 5-10 seconds before use to remove any trapped air bubbles in the solution. An aliquot of 6.7m L of the sample was placed in the sample chamber. The spindle was immersed in the sample and the system allowed to equilibrate to a temperature of 25 °C for a minimum of 30 minutes. The rheometer program set up allowed for the spindle (SC4-18) to increase its shear rate over predefined time intervals and then decrease its shear rate over similar time intervals. The data was automatically logged. A pre-shear was carried out on each sample prior to starting. Two standard viscosity fluids (50 cP and 100 cP) were also measured to ensure rheometer reproducibility. The viscosities measured are only valid for a temperature of 25 °C.

DH analysis

The measurement of the degree of hydrolysis (DH) of the protein was based on the direct quantification of the primary amino groups on the protein with o- phthaldialdehyde (OPA) using serine as the standard (Nielsen, Petersen, & Dambmann, 2001 ). The method was modified for use with 96well-plates. Samples were diluted 20 fold using UltraPure water and reacted with OPA. After reacting for 2 minutes the absorption at 340 nm was measured on a SpectraMax M4 spectrophotometer. All samples were analysed in triplicate.

Protein analysis

The total protein content in the diluted gum samples were determined by the Quant-iT™ assay (Molecular Probes, Life Technologies, USA). Samples were diluted 20 fold in water before testing in triplicate.

Results

Analysis of the thickening properties of the viscosity inhibited concentrate material The functional' viscosity (viscosity of the product post dilution) of the viscosity inhibited concentrate samples from each stage of the process was determined using a Brookfield DV3T Rheometer (see Table 19).

Table 19. Viscosity analysis of the viscosity inhibited concentrate samples.

The viscosities of the samples ranged from 132 ha50cP to 209 ha50cP. Samples CI06, CI07, CI13 and CI14 had similar viscosities (178, 182, 195, 177 ha50cP respectively).

Both the Alcalcase (CI09 and CI10) and the Viscoenzyme ( CI16 and CI17) treated samples had a slight decrease in viscosity compared to their respective controls (151 , 132, 145, 150, 209 and 194 ha50cP, respectively). Determination of the degree of hydrolysis (DH) of the protein in the modified gum material

The protein concentration in each sample is needed in order to calculate the DH after OPA analysis. Although the estimated protein concentration in the samples were provided by the client, these were based on the product specifications of the major components in the starting material. To obtain a more accurate DH value, the actual protein concentration in each sample was determined using the Quant-iT™ method which is known for its greater accuracy over traditional UV assay methods.

The DH of the protein from the starting material through to the final process intermediate was determined using an OPA based method and calculated using both the estimated protein concentration and the value determined by the Quant-iT™ assay (see Table 20).

Table 20. DH of protein in the modified gum samples

* Protein concentration 'estimated' - based on the product specification sheet of starting materia!.

* * Protein concentration 'actual' - total protein content determined by Quant-iT™.

***DH results based on 'estimated' protein concentration. **** DH results based on 'actual' protein concentration.

The DH of the samples ranged from 6.8% to 16.9%. Pre-treatments 1 , 2 and 3 resulted in the largest increase in DH (13.5% for CI04, 14.5% for CI05 and 16.9% for CI08). Papain treatment alone resulted in the smallest increase in DH compared to the starting material (6.8% for CI01 and 8.7% for CI11). Switching the order of enzyme treatment as seen with sample CI12 decreased the DH compared to its counterpart CI05 (11 .9% and 14.5% respectively). Treatment with Viscoenzyme ( CI15) in place of Papain ( CI12) had a decrease in the DH (9% and 11.9% respectively). Conclusion

The proteolytic treatments of the Preblend samples resulted in different levels of DH, however; these were all below 17%. Furthermore, the viscosities of all the viscosity inhibited concentrate samples were within the specifications of an IDDS thickness level 2. This implies that the proteolytic hydrolysis did not change the thickening function of the product. Example 12 - % Composition of a viscosity-inhibited liquid thickener

Below is provided the percentage composition of an embodiment of a viscosity- inhibited liquid thickener demonstrating improved clarity and reduced turbidity when added to clear liquids, such as water.

% Composition of a Clear Instant Liquid Thickener Embodiment