EP4287849A1

EP4287849A1 - Enzyme cross-linked sugar beet pectin microgel particles for use in foods

Info

Publication number: EP4287849A1
Application number: EP22705038.2A
Authority: EP
Inventors: Olivier Jean CAYRE; Isabel CELIGUETA TORRES; FARRES Isabel FERNANDEZ; Aristodimos LAZIDIS; Brent Stuart MURRAY; Samuel Jake STUBLEY; Andrew Steven Whitehouse
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA
Priority date: 2021-02-03
Filing date: 2022-02-03
Publication date: 2023-12-13
Also published as: WO2022167533A1; CN116709932A

Abstract

The invention relates to a method of making sugar beet cross-linked pectin microgel particles. Food products comprising said microgel particles are also provided, for example confectionery products, beverage products, and meat analogue products.

Description

Enzyme Cross-linked Sugar Beet Pectin Microgel Particles for Use in Foods

Introduction

Hydrogels can be defined as 'infinitely' large networks of hydrated polymer molecules which entrap a significant quantity of solvent, either water or electrolyte. In contrast, microgel particles (also referred to as microgels) consist of discrete polymer networks of finite dimensions, swollen by the solvent in which they are dispersed. Similarly to bulk hydrogels, physical and chemical cross-linking can be exploited to control the polymer network swelling depending on the starting materials available and the targeted application.

Most microgels with potential applications in foods consist of supramolecular assemblies of biopolymer molecules, more specifically proteins and polysaccharides. Such biopolymers form gels via intermolecular association under different conditions with heat set, cool set and ionotropic gelation mechanisms being the most common. This implies that the specific technique employed to synthesize microgels is dependent on the chosen biopolymer characteristics.

To be successfully incorporated into commercial formulations, microgel particles must retain their structural integrity throughout any subsequent manufacturing steps, storage and consumer handling. There is a clear need for microgel particles which are more robust, resistant to thermal treatment and show no dissolution on prolonged (several months) storage.

Such microgel particles would have distinct advantages as rheology modifiers as well as useful colloid-stabilizing properties in food products.

Summary of the Invention

The inventors have developed a method of making sugar beet pectin microgel particles which surprisingly addresses the unmet needs above.

The sugar beet pectin microgels of the invention are made by a top-down technique. The microgels have a rheology very different to that of particle dispersions or other biopolymer microgels. A shear thinning response was identified at low shear rates, followed by a Newtonian behavior and a second shear thinning regime on increasing shear rate. This behavior was completely reversible on reducing shear rate, even in the concentrated regime. The absence of hysteresis reflects the robustness of the covalently cross-linked microgel particles, in that their network structure is not irreversibly distorted as they pass each other in the shear field.

These unique properties offer a clear advantage related to their ability to be deposited and recover their texturizing properties at rest in application. Moreover, these microgels are thermally irreversible, resisting dissolution on prolong storage in water-based systems, in contrast to physically cross-linked polysaccharide microgels. This allows their use as an ingredient in food manufacturing processes that have heat treatment steps, in addition to the inline structuring approach.

Embodiments of the Invention

The invention relates in general to a method of making cross-linked pectin microgel particles, preferably cross-linked sugar beet pectin microgel particles.

In a first embodiment, said method comprises a. Developing a covalently cross linked hydrogel from an aqueous solution of sugar beet pectin; and b. Disrupting the hydrogel to form a coarse sugar beet pectin microgel suspension of particles.

In a second embodiment, said method comprises a. Developing a covalently cross linked hydrogel from an aqueous solution of sugar beet pectin; and b. Disrupting the hydrogel to form a coarse sugar beet pectin microgel suspension of particles. d. Adjusting the coarse sugar beet pectin microgel suspension of particles to a desired concentration.

In a third embodiment, said method comprises a. Developing a covalently cross linked hydrogel from an aqueous solution of sugar beet pectin; b. Disrupting the hydrogel to form a coarse sugar beet pectin microgel suspension of particles; c. Disrupting the coarse sugar beet pectin microgel suspension of particles to form a fine sugar beet pectin microgel dispersion of particles; and d. Adjusting the fine sugar beet pectin microgel dispersion of particles to a desired concentration.

In a fourth embodiment, said method comprises a. Incubating an aqueous solution of sugar beet pectin with an enzyme until a covalently cross linked hydrogel develops; b. Diluting the hydrogel in a dispersion medium; and c. Disrupting the diluted hydrogel to form a coarse sugar beet pectin microgel suspension of particles, wherein at least 50% of the volume of the microgel particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction. d. Adjusting the coarse sugar beet pectin microgel suspension of particles to a concentration of at least 0.5 wt% dry weight sugar beet pectin.

In a fifth embodiment, said method comprises a. Incubating an aqueous solution of sugar beet pectin with an enzyme until a covalently cross linked hydrogel develops; b. Diluting the hydrogel in a dispersion medium; and c. Disrupting the diluted hydrogel to form a coarse sugar beet pectin microgel suspension of particles, wherein at least 50% of the volume of the microgel particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction; d. Disrupting the coarse sugar beet pectin microgel suspension of particles to form a fine sugar beet pectin microgel dispersion of particles, wherein at least 50% of the volume of the microgel particles have a size less than 1 micron, as measured by dynamic light scattering; and e. Adjusting the fine sugar beet pectin microgel dispersion of particles to a concentration of at least 0.5 wt% dry weight sugar beet pectin.

The above embodiments can be further described as follows:

Preferably, the aqueous solution of sugar beet pectin is prepared by dispersing sugar beet pectin powder in water. Typically, the powder is allowed to hydrate in the water for a minimum of 12 hours.

Insoluble material may be separated from the aqueous solution by any one of centrifugation, filtration, and sedimentation. The separation method chosen may depend on the sugar beet pectin concentration. At higher concentrations of pectin, the solution will be more viscous and therefore slower and more difficult to filter. In such cases, centrifugation would be preferred because the separation can be done in a shorter time and at a relatively low speed. Preferably, the separation method is centrifugation.

Preferably, the enzyme is a laccase or a peroxidase, for example a horseradish peroxidase enzyme.

Preferably the aqueous solution comprises between 0.5 to 10 wt% sugar beet pectin, preferably between 0.5 to 5 wt%, preferably between 2 to 3 wt%, preferably about 2.4 wt%, sugar beet pectin.

Preferably, a minimum concentration of 0.033 mg laccase enzyme per ml sugar beet pectin aqueous solution is used.

Typically, laccase stock solutions are prepared by solubilizing the enzyme powder in water. The enzyme may be solubilized for a minimum of 20 minutes. Subsequently, laccase can be combined with SBP stock solution via mixing to give a final enzyme concentration of about 0.1 mg ml’¹ laccase.

Preferably, the laccase enzyme has a specific activity of at least 108,000 POU/g (polyphenol oxidase units/gram), preferably a specific activity of about 123,000 POU/g. Typically, the laccase enzyme is from Trametes spp, for example Trametes versicolor.

Preferably, the hydrogel has a shear elastic modulus of at least 10 Pa, preferably 100 Pa to 10 kPA, more preferably about 1 kPa.

More preferably, the aqueous solution comprises between 0.5 to 10 wt%, preferably about 2 wt%, sugar beet pectin, and the hydrogel has a shear elastic modulus of at least 10 Pa.

More preferably, the aqueous solution comprises between 0.5 to 10 wt%, preferably about 2 wt%, sugar beet pectin and a minimum concentration of 0.033 mg laccase enzyme per ml sugar beet pectin aqueous solution is used, wherein the laccase enzyme has a specific activity of at least 108,000 POU/g (polyphenol oxidase units/gram), and wherein the covalently cross linked hydrogel has a shear elastic modulus of at least 10 Pa. Preferably, a gel volume fraction (4>gel) of between 10 to 30%, preferably about 20%, of the hydrogel is diluted in dispersion medium. Preferably, the dispersion medium is water, for example deionized water.

The disruption method to form the coarse sugar beet pectin microgel particles is one which results in irregular and non-spherical shaped particles. Preferably, the disruption method is shear mixing, preferably high shear mixing. Preferably, the disrupting method is rotor stator mixing, for example using a Ultra-Turrax. The gap between rotor and stator may be between 0.2 to 0.4 mm, for example 0.3 mm. The maximum operating speed for preparation of SBP solutions can be between 14,000 to 16,000 rpm, for example about 15,000 rpm. The rotor speed depends on the rotor-stator geometry. Longer and higher speeds (up to 10,000 rpm) does not in general produce smaller particle sizes. Preferably, the coarse sugar beet pectin microgel suspension of particles has a monomodal particle size distribution. Preferably, at least 50 % of the particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction.

The concentration of the coarse sugar beet pectin microgel suspension of particles can be adjusted by centrifugation and subsequent removal of supernatant, for example at about 4000 rpm for 60 to 90 mins.

The total SBP concentration, or CPTOTAL , can be determined by drying in a vacuum oven, for example at 75 °C and a pressure of 600 mm Hg until no change in mass is observed. This allows the accounting for: potential incomplete solubilization of SBP powder, any insoluble material removed by centrifugation and any water associated with the powder before preparing the solutions. The same drying procedure can be used to determine CPTOTAL in the SBP microgel (SBPMG) suspensions prior to their use as emulsifiers.

To allow for a direct comparison between the emulsifying properties of native SBP and SBPMG, solutions or suspensions can be diluted to CPTOTAL = 0.5 wt.%.

The coarse sugar beet pectin microgel suspension of particles can be further disrupted by high pressure homogenization to form a fine sugar beet pectin microgel dispersion of particles. Preferably, greater than 50% of the volume of particles are less than 1 micron as measured by dynamic light scattering. For the most part, they have a non-spherical or irregular morphology. Preferably, the high-pressure homogenization is performed at between 200 to 500 bar, preferably at about 350 bar. Sugar beet pectin coarse microgel dispersion which have been subjected to high pressure homogenization become smaller but, due to having very strong covalent linkage, are still irregular shape particles. Typically, less than 10% of the volume of particles are spherical.

The invention further relates to the fabrication of emulsions, for example oil in water emulsions. The oil in water emulsions may be fabricated according to a method as described herein.

The invention further relates to a covalently cross linked sugar beet pectin microgel particles made by a method according to the invention.

The invention further relates to fine sugar beet pectin microgel particles, wherein said microgel particles have a Z-average hydrodynamic diameter (DH) between 250 to 300 nm, preferably about 279 ± 2 nm, as measured by dynamic light scattering after filtering through a 1 micron syringe.

Microgel particles with a low polydispersity index (PDI) are generally weak and difficult to handle. Furthermore, they may re-dissolve slowly and would be unlikely to form irregular size particles. Preferably, said microgel has a polydispersity index (PDI) between 0.1 to 0.3, preferably about 0.19 ± 0.03.

Preferably, said microgel has a viscosity of about 104 mPa s at a shear rate of 10-3 s-1 in water. Preferably, said microgel has an elastic shear elastic modulus (G') of about 300 Pa after 20 min. Coarser microgels give much higher viscosities at the same concentration of pectin or microgel particles. Viscosities are much higher than simple solutions of the same pectin concentration and also highly reversible in shear.

In general, the pectin based microgels particles of the invention are less prone to microbial attack compared to protein- or starch-based gels. The food preservative used may be, for example, sodium azide, a sorbate salt, benzoate, or natamicine.

Covalently cross-linked pectin microgels of the invention do not re-dissolve in water. This is quite unlike most pectin microgels cross-linked by Ca2+ ions or higher sugar concentrations. Cross-linking through Ca2+ or other multivalent ions leads to leaching of the cross-linking ions over time, particularly when the system is diluted. This weakens the microgels and eventually leads to full dissolution. Chemical cross-linking achieved with laccase enzyme is permanent and so the microgel particles do not re-dissolve.

The invention further relates to a food product comprising a coarse sugar beet pectin microgel suspension of particles or a fine sugar beet pectin microgel dispersion of particles according to the invention.

Preferably, the coarse sugar beet pectin microgel suspension of particles has a monomodal particle size distribution. Preferably, at least 50 % of the particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction.

Perferably, for the fine sugar beet pectin microgel dispersion of particles, greater than 50% of the volume of particles are less than 1 micron as measured by dynamic light scattering. Typically, less than 10% of the volume of particles are spherical.

Preferably, the fine sugar beet pectin microgel particles, have a Z-average hydrodynamic diameter (DH) between 250 to 300 nm, preferably about 279 ± 2 nm, as measured by dynamic light scattering after filtering through a 1 micron syringe.

Preferably, the cross-linked pectin microgels of the invention do not re-dissolve in water.

Preferably the food product is a confectionery product, or a beverage product, or a meat analogue. The confectionery product may be a confectionery filling, for example water based fillings such as fruit fillings, emulsion fillings, and ganache, or that can be applied inside chocolate shells such as tablets, or bonbons, that can be applied inside sugar confections.

The invention further relates to the use of a coarse sugar beet pectin microgel suspension of particles or fine sugar beet pectin microgel dispersion of particles according to the invention, as a rheology modifier or colloid-stabilizer in a food product.

Preferably, the coarse sugar beet pectin microgel suspension of particles has a monomodal particle size distribution. Preferably, at least 50 % of the particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction. Perferably, for the fine sugar beet pectin microgel dispersion of particles, greater than 50% of the volume of particles are less than 1 micron as measured by dynamic light scattering. Typically, less than 10% of the volume of particles are spherical.

Detailed description of the invention

Method of making coarse sugar beet pectin microgel suspension of particles The preferred method of making coarse sugar beet pectin microgel particles comprises preparing an aqueous solution of sugar beet pectin; optionally separating insoluble material from the aqueous solution; incubating the aqueous solution of sugar beet pectin with a laccase or a peroxidase enzyme until a covalently cross linked hydrogel develops; diluting the hydrogel in a dispersion medium; disrupting the diluted hydrogel by high shear mixing to form a coarse sugar beet pectin microgel suspension of particles, wherein at least 50% of the particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction; adjusting the coarse sugar beet pectin microgel suspension of particles to a concentration of between 0.5 to 10 wt% dry weight of sugar beet pectin; and optionally adding food preservative to the coarse sugar beet pectin microgel suspension of particles.

Method of making fine sugar beet pectin microgel dispersion of particles

The preferred method of making fine sugar beet pectin microgel particles comprises preparing an aqueous solution of sugar beet pectin; optionally separating insoluble material from the aqueous solution; incubating the aqueous solution of sugar beet pectin with a laccase or a peroxidase enzyme until a covalently cross linked hydrogel develops; diluting the hydrogel in a dispersion medium; disrupting the diluted hydrogel by high shear mixing to form a coarse sugar beet pectin microgel suspension of particles, wherein at least 50% of the particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction; disrupting the coarse sugar beet pectin microgel suspension of particles, preferably by high pressure homogenization, to form a fine sugar beet pectin microgel dispersion of particles, wherein at least 50% of the particles have a size less than 1 micron as measured by dynamic light scattering; adjusting the fine sugar beet pectin microgel dispersion of particles to a concentration of between 0.5 to 10 wt% dry weight of sugar beet pectin; and optionally adding food preservative to the fine sugar beet pectin microgel dispersion of particles.

Enzyme selection

The preferred enzymes are laccases because they act directly on the substrate via oxidation of ferulic acid esters to phenoxyl radicals. The laccase enzyme may originate from Trametes spp., for example Trametes versicolor. Hydrogen peroxide needs to be added as a source of radicals if peroxidases are used. There is no cross-linking in the absence of hydrogen peroxide. Chemical initiators, for example ammonium or potassium persulphates may be used. They decompose into radical species in water to initiate cross-linking reactions.

Hydrogels

Typically, hydrogels are allowed to develop quiescently, for example under substantially airtight conditions. Typically, hydrogels are allowed to develop for a minimum of 12 hours at 25°C. Preferably, the hydrogel has an elastic modulus between 100 Pa to 10 kPa, more preferably about 1 kPa. Preferably, the hydrogel has a storage modulus of more than 1 order of magnitude greater than the loss modulus. The tan delta of such a hydrogel is less than 0.1. Preferably, the hydrogel has an elastic modulus of at least 100 Pa and a loss modulus of less than 10 Pa.

These hydrogels are the "parent" hydrogels used for the subsequent fabrication of microgel suspensions. The elasticity of SBP hydrogels (and thus microgels subsequently generated from them) depends strongly on the polymer concentration in the gel (C_GEL)-

A hydrogel or microgel can be referred to as "soft" when prepared at C_GEL = 2.4 wt.%.

A hydrogel or microgel can be referred to as "firm" when prepared at C_GEL = 4 wt.%.

Coarse sugar beet pectin microgel suspension For the coarse sugar beet pectin microgel suspension of particles adjusted to a concentration of about 0.8 wt% particles, the relative viscosity is typically at least 25. Typically, concentrated suspensions obtained by centrifugation at 4000 rpm for 60 - 90 mins and discarding of supernatant, particle concentrations up to 1.54% can be obtained. This can be determined through drying a known weight of the suspension in a vacuum oven. Higher concentrations of particles might be achieved, for example by

(i) centrifugation at higher speeds and/or longer times to squeeze water out of discrete, solvent swollen particles; and/or

(ii) evaporating water from the suspensions by application of heat. Water removed may either be from the continuous phase or from discrete particles. Colloidal microgels can be concentrated, for example by rotary evaporation. Typically, concentrations of over 2 wt.% are required before any substantial increase in the relative viscosity is observed.

Typically, at y = 10-3 -0. 1 s-1, shear thinning from 10 Pa s to 1 Pa s is identified. Between y = 0.1 and 100 s-1 the microgel suspension is almost Newtonian. For y > 100 s-1 shear thinning increases again. Typically, at y = 5 x 103 s-1 , //of lOOx that of water was still maintained. This behaviour was completely reversible on decreasing y , even in the concentrated regime. Typically, the covalently crosslinked microgel particles are absent of hysteresis. This reflects the robustness of the covalently cross-linked microgel particles, so that their network structure is not irreversibly distorted even at very high shear rates and particle concentrations, where individual microgel particles are likely squeezed and deformed as they pass each other in the shear field. These unique properties offer advantages relating to their ability to be pumped or deposited and recover their rheology and texturizing properties at rest in application. Typically, the microgels are thermally irreversible. Typically, the microgels resist dissolution in water, particularly in prolonged storage in water-based systems. This is in contrast to physically cross-linked polysaccharide microgels.

Rotor stator mixing

Preferably, the rotor stator mixing speed is 10,000 rpm for about 10 mins. This is because (i) the resulting particle size distribution measured by laser diffraction was found to be monomodal under these conditions. Longer times and higher speeds generally typically result in bimodal particle size distributions; (ii) the resulting particle size (10 to 100 micron) provided suspensions which could easily be concentrated by mild centrifugation and removal of supernatant; and

(iii) sample heating during rotor stator mixing is minimal. There was a risk of viscous heating when higher speeds and longer times were used. Fabrication of oil-in-water (O/W) emulsions

The fabrication of oil in water in emulsions may comprise one or more of the following steps.

Tetradecane can be used as the dispersed phase in the fabrication of O/W emulsions, for example at oil volume fractions (4>_Oii) of c|3 = 20 and 40 % respectively. Typically , the Tetradecane has a p = 0.76 g cm^-3. Typically, the CpTOTAL iS about 0.5 wt.%.

Emulsions can be prepared at 4>_Oji = about 20% for SBP solutions, p can be about 1 g cm'³.

Emulsions can be prepared at 4>_Oji = about 40% for SBPMG suspensions, p can be about 1 g cm'³.

Coarse emulsions can be prepared by combining the O/W phases followed by rotor-stator mixing.

Mixing can be at about 18000 rpm for about 2 min.

Fine emulsions can be prepared immediately by passing the coarse emulsions through a high pressure jet homogenizer, for example at once at about 300 bar. Emulsions can be mixed gently with a vortex prior to decanting into sealed containers and incubating at 25 °C.

Sodium azide (about 0.005 wt%) can be added as a preservative.

Food products

The microgel particles of the invention can be used in food products, for example confectionery products, beverage products, or meat analogue products. Confectionery products, for example confectionery fillings, preferably use microgel particles of D90 particle size between 20 to 80 microns, as measured by static light scattering.

Creamer

Creamers are preferably used for coffee, but can be also used for tea or cocoa, or used with cereals or berries, as a creamer for soups, and in many cooking applications. A liquid creamer of the invention is preferably physically and oxidative stable and overcome phase separation issues for example creaming, plug formation, gelation, syneresis, and sedimentation) during storage at refrigeration temperatures (for example 4 °C), room temperatures (for example 20 °C) and elevated temperatures (for example 30 to 38 °C). The stable liquid creamers can have a shelf-life stability, for example, for at least 9 months, such as at least 6 months at 4 °C and/or at 20 °C, 6 months at 30 °C, and 1 month at 38° C. Beverage composition

A beverage composition may be, for example, a coffee, tea, malt, cereal or cocoa beverage. A beverage composition may be liquid or in powder form. Accordingly, the invention relates to a beverage composition comprising a) a creamer composition of the invention, and b) a coffee, tea, malt, cereal, or cocoa product, for example an extract of coffee, tea, malt, or cocoa. If the beverage composition is in liquid form it may be, for example, packaged in cans, glass bottles, plastic bottles, or any other suitable packaging. The beverage composition may be aseptically packaged. The beverage composition may be produced by a method comprising a) providing a beverage composition base; and b) adding a creamer composition according to the invention to the beverage composition base. By a beverage composition base is understood a composition useful for producing a beverage by addition of a creamer of the invention. A beverage composition base may in itself be suitable for consumption as a beverage. A beverage composition base may be, for example, an extract of coffee, tea, malt, or cocoa.

Definitions

When a composition is described herein in terms of wt%, this means wt% of the total recipe, unless indicated otherwise.

As used herein, "about" is understood to refer to numbers in a range of numerals, for example the range of -30% to +30% of the referenced number, or -20% to +20% of the referenced number, or -10% to +10% of the referenced number, or -5% to +5% of the referenced number, or -1% to +1% of the referenced number.

Hydrogels can be defined as 'infinitely' large networks of hydrated polymer molecules which entrap a significant quantity of solvent, either water or electrolyte.

Microgel particles, also referred to as microgels, can be defined as discrete polymer networks of finite dimensions, swollen by the solvent in which they are dispersed.

The term "coarse" refers to a suspension of particles, wherein at least 50% of the volume of the microgel particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction. The term "fine" refers to a dispersion of particles, wherein at least 50% of the particles have a size less than 1 micron, as measured by dynamic light scattering.

The term "suspension" or "suspension of particles" refers to a non-colloidal dispersed system.

The term "dispersion" or "dispersion of particles" refers to a colloidal system.

The term "sweetener" refers to a mixture of ingredients which imparts sweetness to the final product. These include natural sugars like cane sugar, beet sugar, molasses, other plant derived nutritive sweeteners, and non nutritive high intensity sweeteners.

The term "sugar beet pectin microgel" may refer to a coarse sugar beet pectin suspension of particles or a fine sugar beet pectin dispersion of particles.

The term "thickening agent" may refer to starch, vegetable gum, or flour.

Description of the figures

Figure 1 shows the development of elastic Modulus over time for sugar beet pectin (SBP) hydrogels prepared by the addition of 0.1 mg/ml Laccase Y120. SBP concentration controls gel strength.

Figure 2 shows the conversion of hydrogels of different strength to microgel particles (25 g gel, 100 g water / rotor stator mixing 10,000 rpm, 10 mins). Stronger hydrogels results in larger particles.

Figure 3 shows a comparison of the typical rheological behavior of coarse sugar beet pectin microgels (SBPMG), fine SBPMG, and native SBP solution. The two microgel systems contain the "softest particles" i.e. microgel particles prepared from hydrogels with G' ~ 200-250 Pa.

Figure 4 shows steady shear viscosity curves for 3.75% SBPMG (Coarse) as a function of particle concentration.

Figure 5 shows relative zero-shear viscosity for 3.75% SBPMG (coarse) as a function of concentration. Linear increases are seen at low particle concentrations followed by an exponential increase above a critical concentration, very likely related to volume fraction i.e. viscosity diverges at random close packing (<f>~0.64) for monodisperse hard spheres.

Figure 6 shows particle size distribution (PSD) of coarse (A) and fine (■) SBP microgels as measured by laser diffraction.

Figure 7 shows PSD of the fine SBP microgel dispersion, as measured via DLS following filtration through a 1 pm hydrophillic syringe filter (Zaverage = 279±2nm).

Figure 8 shows the droplet size distributions of 20 vol.% and 40 vol.% tetradecane in water emulsions stabilized by native SBP, soft and firm SBPMG's on day one.

Figure 9 shows the evolution of emulsion droplet size (D₄,₃ / Dg₀) with storage time at 25 °C for 40 vol.% tetradecane in water emulsions stabilized by native SBP, soft SBPMG and firm SBPMG.

Figure 10 shows the viscoelastic moduli as a function of (A) strain amplitude (y) and (B) angular frequency (co) for 40 vol.% tetradecane in water emulsions stabilised by native SBP, soft SBPMG and firm SBPMG.

Examples

Sugar beet pectin (GENU® Beta Pectin), hereafter referred to as SBP, were a generous gift from CP Kelco (Lille Skensved, Denmark). Two different samples of laccase (EC 1.10.3.2) were used: Y120 originating from Trametes spp. was obtained from Amano Enzyme (Nagoya, Japan), hereafter referred to as LAC- Y120 and the other, originating from the fungus Trametes versicolor, was obtained from Sigma Aldrich (Dorset, UK), hereafter referred to as LAC-TV. Type II reverse osmosis water (Suez water purification system, PA, USA) with a minimum resistivity of 18.2 MQ was used throughout.

Example 1

Fabrication of sugar beet pectin (SBP) hydrogels

The SBP concentration was initially optimized so that the resulting hydrogel had a G' being more than 10 fold greater than G". A concentration of 2.4 wt% powder was subsequently chosen. The powder was left to fully solubilize for a minimum of 12 h with magnetic stirring. The SBP solutions were then centrifuged (Eppendorf 5810 R, Stevenage, UK) at 4000 rpm for 20 min in approximately 30 ml aliquots to remove any insoluble material. Following centrifugation, the final SBP concentration of the sample was determined by drying. Laccase stock solutions were freshly prepared by solubilizing the enzyme powder in water for a minimum of 20 min. 25 ml of SBP solution and 5 ml of laccase stock solution (LAC-TV or LAC-Y120) were rapidly combined at ambient temperature, followed by gentle magnetic stirring, to obtain a final SBP concentration of 2 wt.%. When the two solutions were visibly well mixed, the stirrer bar was removed and hydrogels allowed to develop quiescently in sealed containers to reduce solvent evaporation for a minimum of 12 h at 25 °C. Under these conditions, the SBP hydrogels obtained appeared homogenous without any noticeable syneresis.

Example 2

Fabrication of SBP microgel suspensions

SBP microgels were obtained from parent hydrogels prepared by the addition of LAC-TV at 1.2 mg enzyme per ml pectin solution after the 12h storage period. SBP microgel suspensions were fabricated (4>gel = 20% in a deionized water dispersion medium) using a two-step mechanical disruption protocol.

The first step of mechanical disruption was by using a rotor stator mixing device (Model L5MA, Silverson Machines, Buckinghamshire, UK), equipped with a general purpose disintegrating head and operated at 2000 rpm for 60 s. Subsequently, further disruption was induced by passing the dispersion though a high-pressure valve homogenizer (Panda Plus 2000, GEA Niro Soavi Homogenizer, Parma, Italy), pre-set with water to operate 350 bar. The pressure was found to fluctuate slightly with the dispersions but did not exceed 500 bar. Samples were passed through this homogenizer a total of 3 times. Where microgel suspensions were stored for prolonged periods of time, sodium azide was added to at a concentration of 0.005 wt.% to suppress microbial growth.

Samples of the product of the initial rotor stator mixing were also characterized separately, referred to as the "coarse microgel suspension", whilst the product of combined rotor stator mixing and high pressure homogenization is referred to as the "fine microgel dispersion"

Example 3 Enzyme (Laccase) Activity Assay via UV/VIS spectrophotometry

A Specord® 210 PLUS UV/VIS spectrophotometer (Analytik Jena, Jena, Germany) was used to perform enzyme assays in triplicate. The relative activity, at 25 °C, of the LAC-TV and LAC-Y120 were measured by monitoring the oxidation of ABTS substrate via Abs measurements at X = 420 nm for 10 min. Disposable cuvettes with a path length of 1 cm were used. The final reaction volume (3 mL) contained 2 mM ABTS and (10 pL) of enzyme stock solutions, both of which were prepared in Mcllvaine buffer (pH 3).

Example 4

Particle size analysis by laser diffraction

A Mastersizer 3000 equipped with the Hydro EV wet sample dispersion unit (Malvern Instruments, Worcestershire, UK) was used to perform laser diffraction measurements on all pectin microgel samples at ambient temperature (20 °C). The instrument uses two light sources (red light emitted from a 633 nm He-Ne laser and blue light emitted from a 470 nm LED). After optical alignment and measurement of background scattering, microgel samples were added to the dispersion unit until the laser obscuration reached > 1%. Particle size distributions (PSD) were inferred from the angular dependence of scattered light intensity with raw data being modelled in the Mastersizer software using the Mie theory for spherical particles. The Mastersizer dispersion medium was water (refractive index = 1.33). For SBP microgels, a refractive index of 1.35 and an absorption index of 0.01 was used. Representative PSDs are reported alongside mean values of particle diameter, namely the Sauter (surface weighted) mean diameter D3,2 and the volume weighted mean diameter D4,3, calculated according to;

_ SniP a’^b SniD^

Where ni is the number of particles of diameter Di. The width of the PSD is reported in terms of the SPAN;

SPAN = ^DV90~ ^Dv1°

Where Dvx is the diameter of which x percentage of particles are smaller. All values reported are based on the average of 5 measurements on each individual sample.

Example 5

Particle size analysis via dynamic light scattering

A Zetasizer Ultra (Malvern Instruments, Worcestershire, UK) equipped with a He-Ne laser (X = 633 nm) was used to perform dynamic light scattering on the fine microgel suspensions to complement the laser diffraction experiments. The SBP fine microgel suspensions were diluted into deionized water to a concentration of approximately 0.04 wt% and filtered through a 1 pm polyethersulfone hydrophilic syringe filter, into disposable cuvettes for particle size analysis. Measurements were performed in the non-invasive backscatter mode (scattering angle, 9 = 173°) at 20 °C.

The average translational diffusion coefficient, D, was computed in the Zetasizer software by cumulant analysis of the intensity autocorrelation function and subsequently used to calculate the hydrodynamic radius, RH based on the Stokes-Einstein relation:

D =

Where kB is Boltzmann constant, T is the absolute temperature and t] is the viscosity of the solvent. Reported values of samples Z-average hydrodynamic diameter and polydispersity index (PDI) are based on the average of 3 individual measurements, each obtained for a 50 second measurement duration.

Example 6

Solvent removal from SBP microgel suspensions via rotary evaporation

A rotary evaporator (Hei-VAP Advantage, Heidolph Instruments GmbH, Schwabach, Germany) was used to concentrate the fine SBP microgel dispersions for rheological characterization and imaging. Solvent (water) was removed at 35 °C and 200 mbar. The condenser temperature was set to 5 °C using a circulating water bath. The rotational speed used for solvent removal was adjusted during the process to take account of the increasing sample viscosity.

Example 7

Microscopy of SBP microgel suspensions

A Zeiss confocal laser scanning microscope (CLSM) (Model LSM 700, Carl Zeiss Microscopy GmbH, Jena, Germany) was used to image the concentrated fine SBP microgel particles, concentrated to approximately 2 wt.%. The coarse SBP microgels were imaged without concentration. FITC-dextran (0.1 wt. %) was used to stain the continuous phase, dissolved directly into the microgel suspensions. All suspensions were placed in microscope well slides for imaging. For electron microscopy, samples were encapsulated in a 3 wt% agar gel in a plastic tube. The tube contents were fixed by curing for over 8 h in 3.7 wt% formaldehyde solution. Aqueous phase was then replaced by ethanol via successive immersion in baths with increasing ethanol concentration (10 %, 30 %, 50 %, 70 %, 90 % and 100 %). The sample was then dried by immersion in supercritical CO2, followed by release to atmospheric pressure. The sample tubes were cut transversely and the thin sections glued onto an SEM stub and coated with a 10 nm gold layer, then imaged in low vacuum mode via a Quanta F200 Scanning Electron Microscope.

Example 8

Rheology of SBP gelation, SBP solutions and microgel suspensions

An Anton Paar MCR 302 (Anton Paar GmbH, Graz, Austria) rheometer was used at a controlled temperature of 25 °C. Oscillatory shear rheometry was performed on SBP hydrogels prepared as above but using scaled down reaction volumes. Following the combination of SBP and laccase solutions at ambient temperature, the gelling mixture was immediately transferred to the gap between a 50 mm parallel plate measuring set (PP50), with the gap set to 1 mm. Care was taken to avoid overfilling and the sample was trimmed where required. Viscoelastic properties of SBP hydrogels were monitored at 25 °C for up to 60 min using an oscillatory time sweep at a frequency of 1 Hz and a strain amplitude of 0.001 %, which was well within the linear viscoelastic region (LVER) for the final hydrogels. Data points were recorded every 30 s. In some experiments the nominal enzyme concentration (CE) was varied to compare the effect of the 2 different laccases preparations. Viscosity curves for SBP solutions were performed in triplicate using a fresh sample for each measurement and a coneplate measuring set (CP75) with a 1° cone angle and 151 pm cone truncation. After gap setting, the sample was left at rest for 10 min interval for temperature equilibration. Logarithmic ramps through shear rate were used with 5 data points being recorded per decade when in steady state. Roughened parallel plates were used with SBP microgel samples to check for wall-slip effects. A separate PP50 measuring set was roughened by gluing water-resistant silicon carbide sandpaper (600 grit, from 3M) to both the upper and lower plates. The adhesive used was a multi-purpose silicone rubber sealant (Dow Corning 732), cured for a minimum of 12 h before the plates were used. The sample was loaded and the gap set to 400 pm. To erase any mechanical history associated with sample preparation and loading, a pre-shear at a shear rate of 100 s-1 for 60 s was used, followed by a rest period of 45 min before any other measurements commenced. Logarithmic ramps in shear rate were used, from 10’³ to 10³ s’¹, with 5 data points per decade on reaching steady state. All measurement geometries were covered by a custom-made circular plastic hood with dampened kitchen roll fixed to its inner circumference to minimize solvent evaporation and raw data were analyzed in the RheoCompass software (Anton Paar GmbH, Graz, Austria).

Example 9

Sugar beet pectin (SBP) hydrogels

The SBP polymer chains needs to be entangled in order to guarantee efficient gel cross-linking. Therefore, viscosity was measured as a function of polymer concentration to estimate the entanglement concentration regions. At low [SBP] (0.4 and 0.6 wt.%), appeared independent of the shear rate (y ), i.e., Newtonian flow behavior. As the polymer concentration increases, intermolecular interactions become more pronounced and polymer chains will start to interpenetrate, so that by [SBP] = 0.8 wt.%, typical pseudoplastic behavior was seen, due to the progressive disentanglement of polymer chains and their stretching and alignment in the direction of shear at high y , reducing the resistance to flow. At high y , ^ reached an apparent plateau. When the viscosity at an arbitrary y = 0.1 s-1 (or 1 s-1 for 0.4 and 0.6 wt.% particles) was plotted against [SBP], there was consistent evidence that C* is between 0.7 and 1.0 wt%. The [SBP] subsequently used to prepare the hydrogels, from which the microgels were formed, was 2 wt.%, i.e., significantly greater than the estimated value of C*. The nominal enzyme concentration (CE) of LAC-Y120 is only 15% that of LAC-TV, indicating the lower activity of the latter. The assay was also conducted at higher CE and the gradients were also linear (R > 0.997 in all cases) and equal to 1.87, 3.74 and 5.19 x 10-3 s-1 for CE = 0.15, 0.30 and 0.50 mg ml-1 LAC-Y120 laccase, respectively; 1.96, 3.37 and 5.49 x 10-3 s-1 for CE = 1, 2 and 3 mg ml-1 LAC-TV laccase, respectively. LAC-Y120 was found to be 6.1 x more active than LAC-TV across this concentration range.

Example 10

Confectionery microgel filling with improved rheological properties

In step 1 of the batch manufacturing process, SBP microgel particles are created as per Example 2. Once the microgel particles are formed, they are kept at a temperature higher than 30°C for further processing in Step 3.

In step 2 (bulk filling mass), a mixture of sugar and glucose syrup type ingredients (for example inverted sugar syrups, fructose-glucose syrup) (Table 1) are blended under heat (at between 80-110 °C) and medium shear in a jacketed vessel with possibility to heat and cool, for example BCH pan. Other ingredients, such as a fat phase and proteins, may be added instead of the fruit ingredients shown in Table 1, depending on the type of sensory sensation that is desired to be achieved in the final filling. Heating of the mass occurs over time to ensure water evaporation until the final Brix of the mixture is not higher than 90 Brix, preferably 88 Brix, as measured by refractometer. This is to ensure that the water activity (a_w) of the mass is < 0.67 after the equilibrium between the two phases is reached. This is a quality parameter used to ensure a microbiologically stable confectionery filling over shelf life (for between 8-12 months, depending on application) when stored at ambient temperature.

Table 1. In step 3 (mixing the SBP microgel with bulk filling mass), a fraction of SBP microgel from step 1 is mixed with the bulk filling mass of step 2 at a ratio that allows obtaining the desired rheological properties in the final mass. The masses are mixed under low shear, at a temperature not lower than 40°C, to ensure gel particles are homogeneously mixed. The SBP microgel acts as rheology modifier of the filling, allowing to obtain confectionery fillings with different viscosities and thus different sensorial properties.

The final microgel filling can be deposited into chocolate shells (tablets, bonbons) or inside sugar confectionery molds at 28-30°C. The microgel flows and sets upon cooling. Once consumed, it recovers its flowing properties, thus providing a unique sensory sensation.

Example 11

Confectionery microgel filling with improved rheological properties

A continuous approach for creation of microgel confectionery filling is described below using the ingredients in Table 2.

Table 2.

During hydration of pectin, part of the dry sugar content, e.g 2 to 5%, is mixed with the pectin. A portion of the water in the recipe is added to this mixture, which is then heated up to 80°C while stirring to ensure pectin hydration. This blend is reserved, under stirring at this temperature, until further use. If additional ingredients are used in the recipe (for example fruit concentrates, flavors, purees), they are added at this point.

To create a microgel confectionery filling, the remaining sugar containing ingredients (glucose syrup, sucrose), hydrated pectin and water are mixed in a jacketed vessel and heated to 80-110°C, whilst stirring. Laccase enzyme is added to the mixture at approximately 1.2 mg enzyme/mL of pectin. Gelation will initiate while stirring at low shear, and cooling towards 60°C (using cold water applied to the jacketed vessel). At the end of the gelation period, the refractive index of the mass should be below 86 Brix, preferably about 84 Brix, as measured by refractometer. This is to ensure that the water activity (a_w) of the mass is < 0.67, a quality parameter used to ensure production of microbiologically stable confectionery fillings over shelf life of 8 to 12 months, depending on application.

Once the total solids content is as expected, the gel is stirred again and cooled down to approximately 28-30 °C, ready to be deposited into chocolate shell bonbons or into silicone rubber moulds or the like, for example starch moulds, dual shot depositing to create a filled sugar confectionery.

The final microgel filling can be deposited into chocolate shells (tablets, bonbons) or inside sugar confectionery molds at 28-30°C. The microgel flow sets upon cooling. Once it is consumed, it recovers its flowing properties, providing a unique sensory sensation.

Example 12

Creamers made with colloidal SBP microgels and their use in beverages

Colloidal laccase cross-linked SBP microgels are produced using the two step mechanical disruption protocol: SBP hydrogels are prepared by the addition of LAC-TV at 1.2 mg enzyme per ml pectin solution after the 12h storage period. Colloidal SBP microgel suspensions are then fabricated using a rotor stator mixing device (Model L5MA, Silverson Machines, Buckinghamshire, UK), equipped with a general purpose disintegrating head and operated at 2000 rpm for 60 s. Subsequently, further disruption is induced by passing the dispersion though a high-pressure valve homogenizer (Panda Plus 2000, GEA Niro Soavi Homogenizer, Parma, Italy), pre-set with water to operate 350 bar. Samples are passed through this homogenizer a total of 3 times.

Colloidal laccase cross-linked SBP are then used after production in their wet (water suspension) state.

The SBP microgels are used to produce a liquid creamer with enhanced mouthfeel and textural properties when added to a beverage. The method for producing a creamer involves homogenizing an oil and water composition to produce an oil-in-water emulsion. Before homogenization, emulsifiers, proteins, buffers, sweeteners, colloidal SBP microgels and flavors in amounts shown in Table 3 are hydrated in water (at between 40 °C and 90 °C) under agitation with the addition of the melted oil. The composition may be heat treated before by aseptic heat treatment, for example by direct or indirect UHT processes, such as UHT sterilization and UHT pasteurization. Laccase cross-linked SBP microgels resist thermal treatment, hence keeping their functionality as texture and mouthfeel enhancers. An additional benefit of adding the SBP in the form or microgel relates to a greater shear-thinning behavior which is positively associated with lower perception of sliminess compared to when the SBP is added in solution.

Table 3.

When added to a beverage, the creamer produces a physically stable, homogeneous, whitened drink with a good mouthfeel, and body, smooth texture, and a pleasant taste with no off-flavors notes.

Example 13

Stable, texture-enhanced non-dairy plant-based beverages with colloidal SBP microgels

Colloidal, laccase cross-linked SBP microgels can be used to create non-dairy (plant-based) beverages with enhanced mouthfeel and without added hydrocolloid stabilizers to prevent particle (for example cocoa powder) sedimentation. The process consists of pre-hydration of soy protein and optional pre-treatment to 77°C for 3 minutes or reduction of pH to 6.3 combined with heat treatment to 76°C for 3 minutes to partially denature the soy protein. The partial denaturation leads to protein aggregation, providing smoother texture versus the process where soy protein is not pre-treated. The rest of the ingredients shown in Table 4 are then added to the soy protein preparation and mixed. The solution is then pasteurized at 88°C for 25 seconds, and then homogenized at a total pressure of 170 bars.

Homogenization of the whole beverage can be done either prior or after heat treatment, preferably between 120 and 170 bars. The non dairy beverage product is shelf-life stable and has superior organoleptic and textural properties.

The non-dairy beverage made with the added laccase cross-linked colloidal SBP microgels has improved mouthfeel (increased 'body') and does not require the addition of hydrocolloids such as carrageenan or gellan gum to stabilize the cocoa particles against sedimentation.

Table 4.

Example 14

Meat analogue with coarse SBP microgels and coarse SBP emulsion microgels

Coarse laccase cross-linked SBP microgels are produced as follows: SBP hydrogels are prepared by the addition of LAC-TV at 1.2 mg enzyme per ml pectin solution after the 12h storage period. Colloidal SBP microgel suspensions are then fabricated using a rotor stator mixing device (Model L5MA, Silverson Machines, Buckinghamshire, UK), equipped with a general purpose disintegrating head and operated at 2000 rpm for 60 s. The meat analogue can be made by mixing plant extract with the coarse SBP microgel, binding agent, solid fat, and molding, to form a meat analogue. Alternatively, the meat analogue can be made by mixing plant extract with an emulsion gel comprising a binding agent, lipid and the coarse SBP microgel, solid fat, and molding, to form a meat analogue.

In a third variable, a emulsion stabilized by coarse SBP microgel particles can be formed as follows: SBP hydrogels are prepared by the addition of LAC-TV at 1.2 mg enzyme per ml pectin solution after the 12h storage period. Coarse emulsion SBP microgels are then fabricated by adding liquid oil to the previously formed SBP hydrogel and mixing using a rotor stator mixing device (Model L5MA, Silverson Machines, Buckinghamshire, UK), equipped with a general purpose disintegrating head and operated at 2000 rpm for 2 minutes. The coarse emulsion SBP microgels are then mixed with plant extract, binding agent, solid fat, and other ingredients as shown in Table 5, before molding to form a meat analogue. Coarse laccase cross-linked SBP microgels and emulsion microgels provide increased firmness when consumed at temperature of around 60°C due to the thermally stable nature of these laccase cross-linked SBP microgels.

Table 5. Example 15

Emulsions

Oil in water (O/W) emulsions can be stabilized by native SBP or SBPMG at CPTOTAL = 0.5 wt.% and oil (tetradecane) volume fractions of up to 40%. Droplet size can be tailored by controlling the elasticity of the microgel particles. Storage stability against coalescence and Ostwald ripening is improved for SBPMG emulsions due to the presence of particles at the interface. Due to the high energy required to remove particles from the interface, such emulsions remain stable during consecutive heating and cooling cycles demonstrating little difference in their droplet sizes. Typical heating cycles of pasteurization and heating by the consumer. All emulsions remained stable to phase separation after two heating cycles. For 20 vol.% emulsions, creaming stability is lowest for microgel stabilized emulsions due to droplet flocculation which increases the effective particle size and thus the creaming velocity. In the 40 vol.% emulsions, creaming stability is improved for microgel stabilized emulsions which seems to be related to the rheological properties, namely a higher viscosity and elasticity in the presence of particles. The rheological properties however, in turn, arise (in part) due to this flocculation. The effective volume fraction occupied by floccs is greater than freely dispersed droplets (i.e. >40 %) due to the immobilization of solvent in the interstitial regions of the flocc. In addition, one needs to account for the volume fraction occupied by the particles. The reason elasticity is observed is presumably due to either; (i) the development of fractal, branched floccs which span the entire system, analogous to particle gels or (ii) a "soft glassy" or "jammed" structure where particle creaming is retarded due to the immobilization by their nearest neighbors.

Figure 8 shows that the droplet size distribution can be tailored by using different SBP microgel suspensions, firm or soft. In the figure, droplet size distributions of (left) 20 vol.% and (right) 40 vol.% tetradecane in water emulsions are stabilized by native SBP (■), soft SBPMG (A) and firm SBPMG (•). The data was collected within 2 hours of preparation of emulsions. Data for SBPMG emulsions corresponds to "deflocculated" emulsions.

Figure 9 shows that microgel stabilized emulsions can be protected against droplet coarsening on prolonged storage at 25°C and this is due to the particles being irreversibly adsorbed to the interface, creating a thick interfacial film which prevents droplets from coming into contact and thus prevents coalescence. The figure shows the evolution of emulsion droplet size (D₄,₃ / Dg₀) with storage time at 25 °C for 40 vol.% tetradecane in water emulsions stabilized by native SBP (■), soft SBPMG (A) and firm SBPMG (•).

Creaming can be inhibited by increasing the volume fraction of oil, and inclusion of SBP microgels. This creaming was quantified by measuring the creaming index.

The appearance of native SBP stabilised emulsions was assessed before and after exposing the emulsions to two heating and cooling cycles at 75'C and 25'C respectively. Droplet size was clearly seen to increase, which wasn't the case for the microgel stabilised emulsions. The effect of heating emulsions was quantified by laser diffraction. Irrespective of oil volume fraction, the droplet diameters D4,3 and D90 were found to increase only slightly for the microgel emulsions.

Further analysis revealed that by using SBPMG as emulsifiers, the viscosity of the emulsions can be enhanced relative to native SBP solutions. This is in part due to the higher volume fraction of dispersed phase (considering SBPMG's as particulate, space filling objects). There is also potentially an effect of the presence of non-adsorbed microgels which structure the continuous phase. Most likely, the increase in viscosity is simply due to droplet flocculation which acts to increase the volume fraction of dispersed material due to solvent (i.e. continuous phase) being trapped inside the interstitial regions of the floccs.

Figure 10 shows that at 40 vol.%, all emulsions demonstrate elasticity with G'>G". The magnitude of G', the range of frequency (time) and strain (deformation) at which elasticity is observed is greater for the SBPMG stabilized emulsions suggesting that the number and strength of particle/droplet interactions is greater than those in native SBP emulsions. This is again due to the volume fraction of the dispersed phase. Emulsions demonstrate rheological properties typical of a "soft glass" or particle/colloidal gel. In the figure, the viscoelastic moduli (G' = storage modulus = closed symbols, G" = loss modulus = open symbols) as a function of (A) strain amplitude (y) and (B) angular frequency (co) for 40 vol.% tetradecane in water emulsions stabilised by native SBP (■), soft SBPMG (A) and firm SBPMG (•). 1

Claims

1. A method of making sugar beet pectin microgel particles, said method comprising a. Incubating an aqueous solution of sugar beet pectin with an enzyme until a covalently cross linked hydrogel develops; b. Diluting the hydrogel in a dispersion medium; and c. Disrupting the diluted hydrogel to form a coarse sugar beet pectin microgel suspension of particles, wherein at least 50% of the volume of the microgel particles have a size between 1 to 100 microns equivalent spherical diameter as measured by laser diffraction.

2. The method according to claim 1, further comprising disrupting the coarse sugar beet pectin microgel suspension of particles to form a fine sugar beet pectin microgel dispersion of particles, wherein at least 50% of the particles have a size less than 1 micron, as measured by dynamic light scattering.

3. The method according to any previous claim, further comprising adjusting the coarse sugar beet pectin microgel suspension of particles or fine sugar beet pectin microgel dispersion of particles to a concentration of at least 0.5 wt% dry weight sugar beet pectin.

4. The method according to claims 1 to 3, wherein the enzyme is a laccase.

5. The method according to claims 1 to 4, wherein the aqueous solution comprises between 0.5 to 10 wt%, preferably about 2 wt%, sugar beet pectin.

6. The method according to claims 1 to 5, wherein the hydrogel has a shear elastic modulus of at least 10 Pa.

7. The method according to claims 1 to 6, wherein the coarse sugar beet pectin microgel suspension is disrupted using high pressure homogenization at between 200 to 500 bar.

8. A coarse sugar beet pectin microgel suspension of particles made by a method according to claims 1 to 7, or a fine sugar beet pectin microgel dispersion of particles made by a method according to claims 2 to 7.

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9. A food product comprising a coarse sugar beet pectin microgel suspension of particles, or a fine sugar beet pectin microgel dispersion of particles according to claim 8.

10. A food product according to claim 9, wherein said food product is a confectionery product, a beverage product, or a meat analogue product.

11. Use of a coarse sugar beet pectin microgel suspension of particles, or a fine sugar beet pectin microgel dispersion of particles according to claim 8, as a rheology modifier or colloid-stabilizer in food products.

12. Use of a coarse sugar beet pectin microgel suspension of particles, or a fine sugar beet pectin microgel dispersion of particles according to claim 8, as an emulsifier in food products.

13. Use of a coarse sugar beet pectin microgel suspension of particles, or a fine sugar beet pectin microgel dispersion of particles according to claim 8, as an emulsifier in food products.

14. Use according to claim 13, in the absence of any additional thickening agents.