Modulation of textural parameters of plant protein gels

Abstract

The present invention relates to a method for producing a gelation-enhanced plant protein isolate derived from legumes such as mung beans, chickpeas, lentils, and peas. The process involves milling, dry air classification, alkaline extraction with anti-foaming agents, and protein recovery via centrifugation or filtration, optionally followed by spray drying. The protein isolate is enzymatically treated with Glucose Oxidase to improve gelation properties, then further enhanced by calcium lactate and optional natural additives like quillaia extract and glucono delta lactone. The resulting protein isolate contains at least 80% protein on a dry weight basis, forms thermally reversible or irreversible gels, and excludes synthetic crosslinkers, emulsifiers, or animal-derived ingredients, supporting clean-label applications. Additionally, the invention covers methods of incorporating this protein isolate into plant-based food formulations and processing them to yield final products with superior textural attributes including firmness, cohesiveness, elasticity, and mouthfeel.

Description

MODULATION OF TEXTURAL PARAMETERS OF PLANT PROTEIN GELSFIELD OF THE INVENTION

[0001] The present invention relates to a process of modulation of textural parameters of plant protein gels. More particularly, the present invention provides a process for improving the gelation property of plant proteins using glucose oxidase alone and / or in combination with calcium lactate, glucono delta lactone and / or quillaia extract that acts as a gelation enhancer and modulates the textural parameters of plant protein gels.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Over the last two decades, considerable research has focused on improving the functional properties of plant proteins using physical or enzymatic strategies. High-pressure processing, sonication, microwave heating, and irradiation have been investigated, but these methods require capital-intensive equipment and often yield inconsistent results across plant species. Moreover, the regulatory burden and scalability issues have limited the commercial uptake of these methods. In contrast, enzymatic and mineral-based modification approaches offer more food-label- compliant alternatives but remain under-explored for their effect on plant protein gelation in real-world food matrices.

[0004] It is well known in the state of the art that enhancing the gelation properties of the plant proteins holds significant importance for food products. Firstly,enhancing gelation allows for the creation of plant-based food products with improved texture, mouthfeel, and structural integrity, similar to their animal- derived counterparts. The conventional prior arts disclose the plant-based isolates and concentrates that often lack satisfactory gelation capabilities, resulting in suboptimal product attributes such as crumbliness or a gritty texture. Plant-based protein ingredients, such as soy protein isolate, pea protein, and wheat gluten, while widely used, demonstrate formulation-specific weaknesses in gelation. For example, pea protein isolates often result in crumbly textures in high-moisture extrusion systems, while mung bean or lentil proteins form weak or brittle gels when subjected to thermal processing. These issues are compounded by batch variability, poor hydration kinetics, and limited solubility at neutral pH. Such limitations restrict their use in high-functionality food systems where textural mimicry of animal products (e.g., egg, cheese, meat) is essential.

[0005] Thermally induced protein-based gels are formed when the proteins denature and re-form a three-dimensional network upon heating. The factors influencing heat-set gels are crucial for creating stable and desirable gel structures, including the type of protein, amino acids in the protein, pH, temperature, ionic strength, and the presence of certain additives or cross-linking agents. The resulting gel can have different textures, strengths, and functionalities depending on the protein properties and the micro-environment.

[0006] Glucose oxidase (GOx) is an oxidoreductase enzyme that catalyzes the oxidation of glucose into gluconic acid and hydrogen peroxide, thereby enabling oxidative crosslinking of proteins. Glucose Oxidase treatment has been observed to modify gluten proteins (gliadins and glutenin) through the formation of disulfide and non-disulfide crosslinks. Therefore, Glucose Oxidase has been reported for use in breadmaking from various flours including wheat and rice flour (Gujral et al, 2004; Bonet et al, 2006). However, there is no direct reference to the improvement in gelation of plant proteins, more particularly mung beans, chickpeas, lentils and peas.

[0007] EP0963704A2 describes a method for modifying protein-containing food materials, such as wheat flour, fish pastes, poultry and cattle meats, soybean protein, and egg white. The invention involves treating the proteinaceous material with a combination of transglutaminase (TG) and an oxidoreductase, such as glucose oxidase, ascorbate oxidase, or catalase. The treatment aims to improve various properties of the food materials, including gelation potency, shape retentivity, cohesiveness, water-holding capacity, and binding properties. Unlike EP0963704A2, which relies on the combined action of transglutaminase and oxidoreductase on a broad range of animal and plant protein matrices, the present approach is entirely free from transglutaminase and targets the selective oxidation- driven enhancement of gelation in plant proteins, particularly mung bean protein isolates, without the need for synthetic or animal-derived crosslinkers.

[0008] Calcium lactate is known in the state of the art to improve the gelling behavior of animal proteins notably fish protein-based Surimi (Sang et al, 2022). Ultrasound treatments in the presence of calcium lactate improved the gel strength of whey protein isolate (Jiang et al, 2018). Calcium lactate has been an effective coagulant for soy-based tofu. However, none of the prior art discloses the isolated effect of calcium lactate on the gel strength of mung bean or other plant proteins. Calcium lactate has been reported in formulations for improving gelling behaviour in carbohydrates / hydrocolloids such as starch along with cysteine, (Hongsprabhas 2010), gellan (Meng et al., 2013) or sodium alginate (WO2022074217A1).

[0009] Quillaia extract is obtained by aqueous extraction of the milled inner bark or wood of Quillaia saponaria Molina, or other Quillaja tree species. It contains several triterpenoids saponins consisting of glycosides of quillaia acid. Quillaia extracts is currently used as a foaming agent in soft drinks, such as ginger beer, root beer, and cream soda, in cocktail mixes, and as an emulsifier in other foods, such as baked goods, candies, frozen dairy products, gelatine, and puddings. It has been reported to interact with bovine serum albumin and increased the gel strength ofthis animal protein (Kaspchak et al, 2019). However, no prior art discloses its effect on strength of plant protein gels. Glucose oxidase alone and in combination with calcium lactate and / or quillaia extract has not been reported in prior art to modify the gel strength of mung bean protein or any other plant protein. The proposed mechanism of action may involve quillaia saponins facilitating partial unfolding of plant globulins and promoting hydrophobic or hydrogen bonding during gelation, particularly under calcium-rich or oxidative conditions.

[0010] Moreover, conventional prior arts disclosing the technological strategies used to improve gelation of plant proteins involve technological interventions such as high-pressure processing, Ultrasound, Microwave technology (Al Ali et al 2021) and irradiation.

[0011] EP1177725A1 involve specially designed processes that requires a heavy capital investment. However, these are still new technologies and consistency at large scale is yet to be established.

[0012] Another simple strategy used is to modulate the microenvironment of the protein. This is achieved through modulation of pH, charge, addition of gelling agents, modification with cross-linking enzymes like transglutaminases and the like. Most formulations use combinations of ingredients to modulate the gelation property of plant proteins. Polyphosphates or pyrophosphates are mostly used. However, they may be harmful if consumed in large amounts.

[0013] Thus, there remains a critical and unmet need for scalable, cost-effective, and food-label-compliant methods for enhancing the gelation and textural integrity of plant-based protein isolates. In particular, a need exists for solutions that avoid the drawbacks of synthetic cross-linkers, complex equipment, or high-energy processing and yet deliver functional protein matrices capable of mimicking or surpassing animal protein gels in food applications.

[0014] Therefore, the inventor of the present invention has successfully addressed the drawbacks of the existing technology and formulated a process of modulation of textural parameters of plant gels with improved gelation properties.OBJECT OF INVENTION

[0015] It is an object of the present invention to improve the gelation property of plant proteins using Glucose oxidase alone and / or in combination with calcium lactate and / or quillaia extract as a gelation modifier. This enables the improvement in textural properties of plant-based food products. These modified plant based protein are used as an alternative for their animal derived products including but not limited to egg analogues, meat alternatives, dairy analogues and baking ingredients in addition to providing label friendly and healthy ingredients.

[0016] In one aspect, calcium lactate used in combination with Glucose oxidase helps in improving the gelation of plant proteins particularly mung bean protein.

[0017] In another aspect, quillaia extract alone acts as a gel modifier and helps in improving the gelation of plant proteins particularly mung bean protein.

[0018] In yet another aspect, Glucose oxidase used in combination with calcium lactate and quillaia extract also helps in improving the gelation of plant proteins particularly mung bean protein.

[0019] In other aspect, glucono delta lactone is used in combination with Calcium lactate and / or quillaia extract to improve the gelation of plant proteins particularly mung bean protein.SUMMARY OF THE INVENTION

[0020] In one aspect, the present disclosure relates to A method of preparing a gelation-enhanced plant protein isolate for use in plant-based food products, said method comprising:a) milling a plant-based raw material selected from the group consisting of mung beans, chickpeas, lentils, peas, or combinations thereof, into flour having an average particle size optimized for high protein yield and minimal detrimental impact on protein purity; b) fractionating said milled flour via dry air classification to obtain:- a fine fraction enriched in protein; and- a coarse fraction enriched in starch; c) mixing said protein-rich fine fraction with water in a predetermined ratio to form a first slurry; d) adjusting the pH of said first slurry to a value in the range of about pH 8 to pH 10 using an alkali selected from sodium hydroxide, potassium hydroxide, or ammonium hydroxide; e) adding an anti-foaming agent to said first slurry, wherein said antifoaming agent is selected from the group consisting of cetostearyl alcohol, stearates, polydimethylsiloxane, silicone, polyethylene glycol-based compounds, or polypropylene glycol; f) stirring said slurry at ambient temperature for a predetermined time period ranging from 15 minutes to 120 minutes to obtain a proteinrich extract; g) separating starch from said extract via centrifugation using a decanter or equivalent system to yield a supernatant; h) optionally adjusting the pH of said supernatant to about pH 4.0 to pH 5.0 using an acid selected from hydrochloric acid, sulfuric acid, phosphoric acid, or organic acids to precipitate proteins; i) recovering said protein precipitate or protein-rich supernatant by one or more separation techniques selected from centrifugation, microfiltration, ultrafiltration, or nanofiltration to yield a high-purity protein slurry; j) optionally spray-drying the protein slurry to obtain a protein isolate or concentrate in powder form;k) treating the protein slurry or isolate with Glucose Oxidase enzyme at a concentration of 10 to 1000 units per gram of protein (dry weight), in the presence of 0 to 0.05 grams of glucose per unit of Glucose Oxidase, under conditions comprising:- pH between 4.5 and 8.0,- temperature between 15°C and 60°C, and- incubation time ranging from 1 minute to 300 minutes; l) subsequently treating said Glucose Oxidase-treated protein slurry or isolate with Calcium lactate at a concentration ranging from 0.001 to 0.1 grams per gram of protein, followed by mixing for 5 to 30 minutes and pH neutralization; m) optionally treating said protein slurry or isolate with quillaia extract, wherein quillaia extract is present in an amount sufficient to enhance gelation and modulate texture; n) optionally combining said protein slurry or isolate with glucono delta lactone (GDL), in conjunction with or without said Calcium lactate and / or said quillaia extract, to further improve gelation properties; and

[0021] wherein said resulting modified protein isolate exhibits enhanced gelation, structural integrity, and textural performance suitable for use in food applications selected from egg analogues, meat alternatives, dairy analogues, and baking ingredients.

[0022] This invention describes a way to create a plant protein isolate with improved gelation properties, ideal for use in plant-based foods like egg substitutes, meat alternatives, and dairy-free products. Starting with plant materials such as mung beans, chickpeas, lentils, or peas, the raw ingredients are milled and then separated to isolate a protein-rich fraction. This fraction is mixed with water, treated with an alkaline solution and anti-foaming agents, and stirred to extract the protein. After removing starch and recovering the protein, the product can be spray-dried into powder form if needed.

[0023] A key step is treating this protein with the enzyme glucose oxidase, which strengthens its gel-forming ability. This is followed by adding calcium lactate and optionally natural ingredients like quillaia extract and glucono delta lactone to further enhance texture and gel strength.

[0024] The resulting protein isolate is high in protein (at least 80%) and forms strong gels when heated and hydrated — without relying on synthetic additives or animal-derived ingredients — making it a clean-label option.

[0025] Finally, the invention includes using this protein isolate in food formulations, which are then processed by heating, acidification, enzymatic action, or calcium crosslinking to produce plant-based foods with better texture, firmness, and mouthfeel.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary embodiments and, together with the description, serve to explain the disclosed principles:Figure la illustrates an image of a preparation of a modified plant protein gel with enhanced textural properties comprising Sample A (Calcium lactate + glucose oxidase).Figure lb illustrates an image of a preparation of a modified plant protein gel with enhanced textural properties comprising Sample B (Calcium lactate + Quillaia Extract).Figure 1c illustrates an image of a preparation of a modified plant protein gel with enhanced textural properties comprising Sample C (Glucono delta lactone (GDL), Calcium lactate, Quillaia extract).Figure Id illustrates an image of a preparation of a modified plant protein gel with enhanced textural properties comprising Sample D (Control mung bean protein).Figure 2a illustrates evaluation of the Sample A for gelation by Rheology.Figure 2b illustrates evaluation of the Sample B for gelation by Rheology.Figure 2c illustrates evaluation of the Sample C for gelation by Rheology.Figure 2d illustrates evaluation of the Sample D for gelation by Rheology.DESCRIPTION OF THE INVENTION

[0027] In the description that follows, a number of terms are used, the following definitions are provided to facilitate understanding of various aspects of the disclosure. In the specification, the word “comprising” is used as an open ended term, substantially equivalent to phrase “including, but not limited to,” and the word comprises has a corresponding meaning.

[0028] The terms and words are used in the following description are not limited to the bibliographical meanings, but, are merely used to enable a clear and consistent understanding of the disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present disclosure are provided for illustration purpose only and not for the purpose of limiting the disclosure as defined by the appended claims and their equivalents.

[0029] In describing the embodiment of the invention, specific terminology is chosen for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that such specific terms include all technical equivalents that operate in a similar manner to accomplish a similar purpose. As used herein, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements.

[0030] The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the inventions are indicated. Indeed, the invention may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.

[0031] The present invention in one aspect, envisages a method of preparing a gelation-enhanced plant protein isolate from raw pulse-based plant material. The term "gelation-enhanced" as used herein refers to the improved capacity of the protein isolate to undergo gelation under appropriate conditions, forming either thermally reversible or irreversible gel matrices that exhibit increased gel strength, cohesiveness, elasticity, and structural integrity. This functionality is crucial for plant-based food systems mimicking the role of eggs, dairy proteins, or gelatin in traditional food products. The plant-based products which uses said plant protein isolate are used as an alternative for their animal derived products such as egg analogues, meat alternatives, dairy analogues and baking ingredients and additionally provides label friendly and healthy ingredients.

[0032] In accordance with an embodiment of the present invention, the method of modulation of textural parameters of plant protein gels comprising the steps of: Milling raw material (mung bean) into specific particle size optimized for higher protein yield; dry fractionating using air classifier to obtain fine protein rich fraction and coarse starch rich fraction; mixing said fraction with water in pre-determined ratio to obtain slurry; adjusting said slurry to pre-determined pH using a alkali and stirring for pre-determined time period to obtain an extract; subjecting said extract to separation of starch using a decanter by centrifugation to obtain supernatant, precipitating protein by adjusting pH of said supernatant using an acid; adding an anti-foaming agent to said slurry and stirring for pre-determined time period to obtain an extract; separating said protein precipitate using a combination of centrifugation and / or micro, ultra or nano filtration to obtain high purity protein slurry; treating said protein slurry for gelation process and spray drying said protein slurry to obtain protein isolate or concentrate.

[0033] In one embodiment of the present invention, disclosed is the method of preparing a gelation-enhanced plant protein isolate for use in plant-based food products. The method comprises the following steps:Step a: Milling of Raw MaterialThe first step of the method involves milling a raw plant-based material selected from the group consisting of mung beans, chickpeas, lentils, peas, or combinations thereof. The raw material may optionally undergo pre-processing steps such as cleaning, sorting, soaking, drying, and dehulling to improve the downstream process efficiency and protein recovery.Milling is conducted using one or more suitable milling devices, including but not limited to hammer mills, pin mills, burr mills, or air classification mills. The objective of this step is to obtain a finely ground flour wherein the average particle size is optimized to below 200 microns, preferably between 50 and 150 microns. This particle size distribution facilitates efficient air classification and enhances the exposure of intracellular protein bodies for subsequent extraction, without causing excessive mechanical shear that could degrade protein integrity.Step b: Dry Air ClassificationThe milled flour is subjected to air classification using a dry fractionation apparatus that leverages differences in particle size and density to separate the flour into two fractions:• A fine fraction that is enriched in protein and typically comprises smaller, less dense particles.• A coarse fraction that is enriched in starch and fibrous material.Air classification is conducted at carefully controlled airflow rates, rotor speeds, and classifier configurations tailored to the specific botanical origin of the feedstock. The protein-rich fine fraction is recovered for subsequent aqueousextraction, while the starch-rich coarse fraction may be discarded, reprocessed, or used for other applications.Step c: Slurry PreparationThe fine protein-rich fraction is mixed with deionized or filtered water in a predetermined solid-to-liquid ratio, typically in the range of 1 :5 to 1 :15 (w / v), to form a homogeneous slurry. Mixing is performed under mild agitation using mechanical stirrers, overhead impellers, or static mixers, ensuring full hydration of proteinaceous material.Step d: pH AdjustmentThe pH of the slurry is adjusted to an alkaline range, preferably between pH 8.0 and pH 10.0, using a food-grade alkali selected from sodium hydroxide (NaOH), potassium hydroxide (KOH), or ammonium hydroxide (NH4OH). This alkalization facilitates solubilization of storage proteins and disrupts protein-starch-lipid complexes, thereby enhancing protein extraction efficiency.The pH adjustment may be conducted in a gradual manner using titration pumps or manual addition, with constant pH monitoring using a calibrated probe to ensure stability. The selected pH must also be tailored to avoid irreversible protein denaturation while maximizing solubility.Step e: Anti-Foaming Agent AdditionDue to the formation of surface-active peptides and protein-polysaccharide complexes, foaming during slurry mixing can be problematic and lead to protein loss and reduced filtration throughput. To address this, an anti-foaming agent is added to the slurry, selected from the group consisting of cetostearyl alcohol, stearates, polydimethylsiloxane, silicone emulsions, polyethylene glycol-based surfactants, or polypropylene glycol.The concentration of anti-foaming agent is optimized to ensure maximum suppression of foam with minimal interference in downstream processing. In a preferred embodiment, polypropylene glycol is used at a concentration of 0.05% to 0.2% (w / v).Step f: MixingThe slurry is then stirred at ambient temperature (20-25°C) for a predetermined time ranging from 15 minutes to 120 minutes, preferably around 60 minutes, to allow for complete extraction of soluble proteins. The agitation is performed under low to moderate shear conditions, which minimizes protein unfolding or aggregation while maximizing diffusion of soluble components into the aqueous phase.Step g: Starch Separation via CentrifugationFollowing extraction, the slurry is subjected to a separation process to remove insoluble starch, fiber, and other coarse debris. This is typically achieved via centrifugation using a decanter centrifuge, disc-stack centrifuge, or equivalent system that operates on principles of sedimentation under high centrifugal force.Parameters such as rotor speed (typically 3,000 to 6,000 rpm), flow rate, residence time, and temperature are carefully controlled to ensure maximum recovery of the protein-rich supernatant while minimizing cross-contamination with starch or non- proteinaceous components.The resultant supernatant contains dissolved or colloidally suspended proteins, residual soluble carbohydrates, and micronutrients, whereas the pellet comprises sedimented starch granules, fibrous particulates, and residual insoluble material.Step h: Optional Isoelectric PrecipitationIn an optional step, the supernatant may be subjected to isoelectric precipitation to recover proteins in solid form. This involves lowering the pH of the protein-richsupernatant to approximately 4.0 to 8.0 using a food-grade acid such as hydrochloric acid (HC1), sulfuric acid (H2SO4), phosphoric acid (H3PO4), citric acid, lactic acid, or other organic acids. The pH chosen typically corresponds to the isoelectric point of major globulin and albumin fractions in the pulse protein matrix.At the isoelectric point, proteins lose their net surface charge, aggregate, and precipitate from solution. The resulting protein curd can be separated by additional centrifugation or filtration. In certain embodiments, this precipitation step may be omitted in favor of retaining a soluble protein fraction with desirable functionality.Step i: Recovery of Protein SlurryThe precipitated protein or directly extracted protein solution is then subjected to advanced separation techniques to purify and concentrate the protein content. The separation techniques may include:• Centrifugation for coarse removal of non-protein particulates;• Microfiltration (MF) for size-exclusion of larger polysaccharides and microbial contaminants;• Ultrafiltration (UF) using membranes with a molecular weight cut-off (MWCO) of 10-50 kDa to retain globular proteins;• Nanofiltration (NF) for partial demineralization and removal of low molecular weight solutes.The combination of UF and NF in tandem allows for refinement of the protein slurry to a target purity level of >80% protein on a dry weight basis. Membrane material, flow rates, pressures, and temperatures are optimized based on the botanical origin and initial protein profile.Step j: Optional Spray-DryingThe resulting high-purity protein slurry may optionally be spray-dried using atomization dryers operated at inlet temperatures of 140-190°C and outlettemperatures of 60-85°C. Spray drying converts the slurry into a flowable powder, which constitutes the protein isolate or concentrate. This form provides enhanced shelf stability, portability, and ease of formulation.However, in embodiments where enzymatic or other gelation-enhancing treatments are to be applied, the liquid slurry is retained for further modification.Step k: Enzymatic Crosslinking with Glucose OxidaseThe core transformation step of the method involves enzymatic treatment with Glucose Oxidase (GOx), an oxidoreductase enzyme that catalyzes the oxidation of glucose to gluconic acid, concurrently generating hydrogen peroxide (H2O2). The generation of H2O2 is exploited to induce protein crosslinking through oxidation of sulfur-containing and aromatic amino acids, especially cysteine and tyrosine residues.The Glucose Oxidase is added at a concentration of 10 to 1000 enzymatic units per gram of protein (dry basis), in the presence of glucose at 0 to 0.05 grams per unit of enzyme. The enzymatic reaction is carried out under tightly regulated conditions:• pH: 4.5 to 8.0, preferably pH 6.0;• Temperature: 15°C to 60°C, preferably around 37°C;• Time: 1 to 300 minutes, optimally 90 minutes.This enzymatic system enhances the protein’s gel-forming capacity via intra- and inter-molecular linkages without the need for synthetic additives.Step 1: Mineral Crosslinking with Calcium LactateFollowing Glucose Oxidase treatment, the protein slurry or isolate is subjected to ionic crosslinking using calcium lactate, which introduces divalent calcium ions capable of forming electrostatic bridges between negatively charged carboxyl groups on protein backbones.Calcium lactate is added in an amount ranging from 0.001 to 0.1 grams per gram of protein, and the mixture is stirred for 5 to 30 minutes, preferably 15 minutes, at room temperature. A subsequent pH neutralization step ensures stabilization of the crosslinked protein matrix.This treatment imparts additional network rigidity, improving the viscoelastic and textural attributes of the protein.Step m: Optional Addition of Quillaia ExtractIn certain embodiments, the gelation-enhanced protein is further treated with quillaia extract, a natural surfactant-rich botanical derivative from Quillaja saponaria. Quillaia extract contains saponins that facilitate protein unfolding, emulsification, and interfacial structuring.It is added in concentrations of 0.01% to 1% (w / w) relative to the protein content, depending on desired functional outcomes. This treatment further modulates the gelation threshold, gelling kinetics, and mouthfeel properties of the protein.Step n: Optional Addition of Glucono Delta Lactone (GDL)As an optional or synergistic step, glucono delta-lactone (GDL) may be added. GDL hydrolyzes in aqueous solution to slowly release gluconic acid, thereby inducing gradual acidification. This enables controlled in situ gelation, particularly useful in applications mimicking set-style yogurt or egg curds.GDL is added in amounts of 0.05% to 0.5% (w / w), either alone or in combination with calcium lactate and / or quillaia extract. This approach allows for programmable texture formation in the final food matrix.Step o: Functional Attributes of Final Protein IsolateThe resulting protein isolate, whether in slurry or powder form, exhibits enhanced gelation characteristics, including:• Increased gel strength (>200 g / cm2, measured via texture analyzer);• Greater thermal and pH stability;• Superior water-holding capacity and cohesiveness;• Minimal syneresis upon heating or cooling;• Suitability for incorporation into diverse food matrices such as egg analogues, cheese alternatives, plant-based meats, and baking formulations.The product is free from synthetic emulsifiers, gums, or animal-derived components and is compliant with clean-label, vegan, and allergen-reduction standards.

[0034] In one exemplary embodiment of the present invention, the protein source is milled into flour. Also, during the milling step of the method the average particle size of said protein extract is reduced to achieve high yield without having any detrimental impact on the purity of the protein isolate.

[0035] In accordance with an embodiment of the present invention, the precipitation of protein is carried out by adjusting the pH of said supernatant using an acid.

[0036] In yet another exemplary embodiment of the present invention, the precipitation technique is an isoelectric precipitation. The pH is adjusted to 4.5 using 5N HC1 for a time period of one hour. However, the isoelectric precipitation step is optional, and protein can be directly separated from the supernatant thus saving on process time and cost.

[0037] In still another exemplary embodiment of the present invention, antifoaming agent is added to said first slurry followed by stirring for pre-determined time period to obtain an extract. In another embodiment first slurry is stirred for 1 hour at room temperature after adding anti-foaming agent. The anti-foaming agent is added to the slurry to control the foaming during the mixing process thus leading higher yield of proteins.

[0038] In yet another exemplary embodiment of the present invention, the first slurry is stirred for 1 hour at room temperature after adding an anti-foaming agent. The anti-foaming agent is added to the slurry to control the foaming during the mixing process thus leading higher yield of highly functional proteins. The use of anti-foaming agent and temperature conditioning owing to the freezing step enable high recovery of highly functional proteins.

[0039] In an exemplary embodiment of the present invention, the protein isolate is treated with Glucose Oxidase lOunits / gm to 1000 units / gm of protein on dry basis, 0-0.05g glucose per unit of glucose oxidase at a pH of 4.5-8 and incubated for 1- 300 min at temperature of 15 - 60 °C.

[0040] In accordance with another exemplary embodiment of the present invention, the protein isolate undergoes Calcium lactate treatment wherein Calcium lactate is added at a dosage of 0.001- 0.1g per gm protein, mixed well for lOmin at room temperature and slurry is neutralized.

[0041] In accordance with yet another exemplary embodiment of the present invention, the method comprises plant protein source which may be prepared from any suitable source of pulse protein, where the starting material is whole plant material (e.g., whole mung bean). In some such embodiments, raw pulse protein materials (e.g., mung beans) may be de-hulled in one or more steps of pitting, soaking, and drying to remove the seed coat (husk) and pericarp (bran). The dehulled materials (e.g., mung beans) are then milled to produce a composition e.g., flour including pulse flours, de-oiled / defatted oilseed flours / cereals with a well- defined particle distribution size. The types of mills employed may include one or a combination of a hammer, pin, knife, burr, and air classifying mills.

[0042] In accordance with the present invention, the protein i sol ate / concentrate obtained from the said method has enhanced gelation properties that allows for the creation of plant-based food products with improved texture, mouthfeel, and structural integrity, similar to their animal-derived counterparts. By enhancing gelation, food technologists can create plant-based alternatives that closely mimicthe sensory experience of meat and dairy products, thereby appealing to a broader consumer base. Furthermore, improved gelation can facilitate the use of plant proteins for replacing egg and whey protein in bakery products, contributing to the sustainability and healthiness of the food systems. Consequently, with the improvement of the gelation property of plant proteins, the textural properties of plant-based food products are also modulated.

[0043] Examples and Experimental ValidationTo further elucidate and validate the method of preparing a gelation-enhanced plant protein isolate, the following examples are provided. These examples are intended to illustrate specific embodiments of the invention and demonstrate the superior gelation and textural performance of the modified protein isolates.

[0044] Preparation of a Modified Plant Protein Gel with Enhanced Textural PropertiesProcedure 1: Sample A (Calcium lactate + glucose oxidase) and depicted in Figure 1AObjective:To illustrate a method for modulating the textural parameters of mung bean protein (MBP) gel by incorporating calcium lactate and enzymatic treatment.Procedure:1. 200 grams of mung bean flour were dispersed in 2000 millilitres of distilled water under continuous stirring. The pH of the slurry was adjusted to 9 using a food grade alkali solution. The mixture was incubated for 1 hour at room temperature under agitation to ensure adequate protein solubilization.2. The mixture was subjected to centrifugation. The supernatant containing solubilized proteins was collected.3. The pH of the supernatant was adjusted to 4.5 using a food grade acid to induce protein precipitation. The mixture was incubated for 1 hour at room temperature to allow complete protein aggregation.4. The precipitated proteins were recovered by centrifugation. The resulting protein-rich pellet was designated as the "protein cake."5. The pH of the protein cake was adjusted to 5.0. Enzymatic hydrolysis was performed by adding 7680 units of the enzyme and 0.000623 grams of dextrose per unit of Glucose oxidase to the protein cake. The mixture was blended for 30 minutes to ensure homogeneity and then incubated at 30°C for 1 hour to initiate enzymatic modification.6. Following enzymatic incubation, 0.57g of calcium lactate was added. The mixture was stirred for 15 minutes to incorporate the gelation agent uniformly.7. The pH was adjusted to 6.5-7 pH using alkali.8. The modified protein gel was freeze-dried to obtain a powder.Procedure 2: Sample B (Calcium lactate + Quillaia Extract) and depicted in Figure IB(Quillaia extract as described in FAO JECFA Monographs (Typel or Type2) is available commercially as liquid product or as spray-dried powder that may contain carriers such as lactose, maltitol or maltodextrin. The liquid product is usually preserved with sodium benzoate or ethanol. Examples include SuperSap, Natural Response, Chile; Quil-A, Superfos, Denmark, Q-Natural 100V, Q-Natural 200V or similar)1. The protein cake is prepared as per procedure 1 (steps 1 to 4). The pH of the protein cake was adjusted to 5.0. Enzymatic hydrolysis was performed by adding 7680 units of the enzyme and 0.000623 grams of dextrose per unit of Glucose oxidase. The mixture was blended for 30 minutes to ensurehomogeneity and then incubated at 30°C for 1 hour to initiate enzymatic modification.2. Following enzymatic incubation, 0.57g of calcium lactate and Quillaia Extract is added at level of 0.14g / litre saponin concentration. The mixture was stirred for 15 minutes to incorporate the gelation agents uniformly.3. The pH was adjusted to 6.5-7 pH using alkali.4. The modified protein gel was freeze-dried to obtain a powder.Procedure for making Sample C-Glucono delta lactone (GDL), Calcium lactate, Quillaia extract and depicted in Figure 1C1. 200 grams of mung bean flour were dispersed in 2000 millilitres of distilled water under continuous stirring. The pH of the slurry was adjusted to 9 using a food grade alkali solution. The mixture was incubated for 1 hour at room temperature under agitation to ensure adequate protein solubilization.2. The mixture was subjected to centrifugation. The supernatant containing solubilized proteins was collected.3. The pH of the supernatant was adjusted to 5.8 using a 4% Glucono delta lactone solution to induce protein precipitation followed by further reduction in pH to 5 using IM HCL. The mixture was incubated for 1 hour at room temperature to allow complete protein aggregation.4. The precipitated proteins were recovered by centrifugation. The resulting protein-rich pellet was designated as the "protein cake."5. Enzymatic hydrolysis was performed by adding 7680 units of the enzyme and 0.000623 grams of dextrose per unit of Glucose oxidase. The mixture was blended for 30 minutes to ensure homogeneity and then incubated at 30°C for 1 hour to initiate enzymatic modification.6. Following enzymatic incubation, 0.57g of calcium lactate was added. The mixture was stirred for 15 minutes to incorporate the gelation agents uniformly.7. The pH was adjusted to 6.5-7 pH using alkali.8. The modified protein gel was freeze-dried to obtain a powder.Procedure for making Sample D (Control mung bean protein) and depicted in Figure ID1. 200 grams of mung bean flour were dispersed in 2000 millilitres of distilled water under continuous stirring. The pH of the slurry was adjusted to 9 using a food grade alkali solution. The mixture was incubated for 1 hour at room temperature under agitation to ensure adequate protein solubilization.2. The mixture was subjected to centrifugation. The supernatant containing solubilized proteins was collected.3. The pH of the supernatant was adjusted to 4.5 using a food grade acid to induce protein precipitation. The mixture was incubated for 1 hour at room temperature to allow complete protein aggregation.4. The precipitated proteins were recovered by centrifugation. The resulting protein-rich pellet was designated as the "protein cake."9. The pH was adjusted to 6.5-7 pH using alkali.10. The modified protein gel was freeze-dried to obtain a powderEvaluation of the samples for gelation by using the Gel Scoring System: Each of the protein powders were reconstituted in water to form a solution of 15% protein solution. The solution was stirred for 30 min at room temperature. The solution was then placed in a water bath at 90degC for an hour. It is cooled for 30mins at room temperature and the formed gel is evaluated by a scoring system.To evaluate the structural integrity and firmness of the resulting protein gels, a qualitative scoring system was employed, ranging from (+) to (++++):(+) - Paste or semi-formed liquid(++) - Weak gel structure; minimal firmness, high syneresis observed(+++) - Moderately weak gel; holds shape briefly but yields readily under slight pressure(++++) - Intermediate gel; retains shape and exhibits moderate elasticity(+++++) - Firm gel structure; good water retention and elasticity, minimal syneresisResults:The gel scoring assessment demonstrates the effectiveness of the applied textural modification techniques in enhancing plant protein gel structure. Sample D, which served as the control (no enzymatic or gelation treatment), exhibited a minimal gel score of (+), indicating poor structural integrity, high syneresis, and weak cohesiveness. This benchmark highlights the baseline gel-forming ability of the untreated mung bean protein.In contrast, Samples A, B, and C, which underwent specific process modifications, showed significant improvement:Sample A achieved the highest gel score of (+++++), indicating a very firm, elastic, and cohesive gel with excellent water-holding capacity and shape retention.Samples B and C both scored (+++), representing moderate gel structures with acceptable firmness and reduced syneresis compared to the control.The progressive enhancement from Sample D to Sample A underscores the impact of enzymatic treatment and gelation agent incorporation on protein networkformation. Among the formulations tested, all the treatments demonstrated superior textural properties, compared to the control validating its suitability for structured plant-based food applications.

[0045] Evaluation of the samples for gelation by Rheology Gelation of the mung bean protein isolates was characterized by dynamic oscillatory rheology. A rheometer (MCR502, Anton Paar) equipped with a bob and cup geometry was used. The rheology protocol comprised a conditioning step at 20DegC for 5 minutes where Amplitude= 1 % and oscillation Frequency = 0.1 Hz. This was followed by a heating step with temperature ramp of 20 deg C to 95 deg C @5deg / min. The temperature was held at 95 deg C for 60minutes, followed by cooling from 95degC to 20degC @5deg / min, and held at 20degC for 5minutes. The Amplitude = 1% and Oscillation Frequency = 0.1 Hz. Samples of mung bean protein isolates were prepared in distilled water at 15% protein concentration. The graphical representation of the results are illustrated in Figures 2A to 2D respectively.ResultsA) Initial Rheological Interpretation at 20°C (Pre-Heating Phase)This phase reveals the viscous / elastic behaviour of each sample before any thermal treatment. Sample B, shows a viscous behaviour, just like the Sample D (control sample). Sample A and C display weak elastic behaviour. This could be anindication of enzymatic crosslinking playing a role. Sample C in which Glucose oxidase, calcium lactate and Quillaia all three modifiers were present is have the highest storage modulus(G’) and also the highest difference between the storage(G’) and loss modulus (G”).B) Rheological Behaviour of Plant Protein Gels at 95°C constant holding after temperature rampThe data compares the storage modulus (G') and loss modulus (G") of four mung protein-based formulations under thermal conditions (95°C). Sample D, the untreated control, shows minimal gel strength with G' peaking below 23 Pa. In contrast, Sample A demonstrates the highest elasticity with G' values exceeding 2000 Pa, indicating a dense and heat-stable network. Sample B and Sample C also indicates strong gelation with high G' and moderate G" values. All the treatments have shown a positive impact on improving the gelation compared to the control mung bean protein.C) Rheological Behaviour of Plant Protein Gels at 20°C constant holding after coolingThis phase evaluates each protein sample’s ability to rebuild gel structure upon cooling after being subjected to thermal stress. Data for Sample A indicates highest structural stability and post-thermal elasticity upon cooling as indicated by the G7G" ratio of greater than 5X. Sample B is also indicated to have good structure with a slightly more elastic structure. Sample C exhibits moderate recovery with G7G" ratio of ~4.5-5.0. Sample D fails to recover meaningfully, validating its role as a negative control and highlighting the need for targeted modification.Thermal reversibility is indicated when the storage modulus (G') and loss modulus (G") decrease upon cooling, suggesting that the gel transitions back toward a more fluid-like state; conversely, a continued increase or stabilization of G' during cooling reflects thermal irreversibility, where the gel network remains permanently set and does not return to its pre-heated, less structured form.Data table for G’ and G” at 20°C constant holding after coolingSamples A, B and C gels are thermally irreversible. Sample D remains mostly unstructured, and does not qualify as thermo-reversible either.

[0046] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. The disclosures and the description herein are intended to be illustrative and are not in any sense limiting the invention. Many changes, modifications, variations and other uses and applications of the subject invention will become apparent to those skilled in the art after considering this specification, which disclose the preferred embodiments thereof. All such changes, modifications, variations and other uses and applications, which do not depart from the spirit and scope of the invention, are deemed to be covered by the invention.

[0047] While, this provisional patent application contains the description of the principal inventive concepts, the complete patent application pursuant hereto, will fully and particularly describe the preferred embodiments of the present invention.

Claims

CLAIMS:

1. A method of preparing a gelation-enhanced plant protein isolate for use in plant-based food products, said method comprising: a) milling a plant-based raw material selected from the group consisting of mung beans, chickpeas, lentils, peas, or combinations thereof, into flour having an average particle size optimized for high protein yield and minimal detrimental impact on protein purity; b) fractionating said milled flour via dry air classification to obtain:- a fine fraction enriched in protein; and- a coarse fraction enriched in starch; c) mixing said protein-rich fine fraction with water in a predetermined ratio to form a first slurry; d) adjusting the pH of said first slurry to a value in the range of about pH 8 to pH 10 using an alkali selected from sodium hydroxide, potassium hydroxide, or ammonium hydroxide; e) adding an anti-foaming agent to said first slurry, wherein said antifoaming agent is selected from the group consisting of cetostearyl alcohol, stearates, polydimethylsiloxane, silicone, polyethylene glycol-based compounds, or polypropylene glycol; f) stirring said slurry at ambient temperature for a predetermined time period ranging from 15 minutes to 120 minutes to obtain a proteinrich extract; g) separating starch from said extract via centrifugation using a decanter or equivalent system to yield a supernatant; h) optionally combining said supernatant with glucono delta lactone (GDL) followed by adjusting the pH to about pH 4.0 to pH 6.0 using an acid selected from hydrochloric acid, sulfuric acid, phosphoric acid, or organic acids to precipitate proteins; i) recovering said protein precipitate or protein-rich supernatant by one or more separation techniques selected from centrifugation,microfiltration, ultrafiltration, or nanofiltration to yield a high-purity protein slurry; j) optionally spray-drying the protein slurry to obtain a protein isolate or concentrate in powder form; k) treating the protein slurry or isolate with Glucose Oxidase enzyme at a concentration of 10 to 1000 units per gram of protein (dry weight), in the presence of 0 to 0.05 grams of glucose per unit of Glucose Oxidase, under conditions comprising:- pH between 4.5 and 8.0,- temperature between 15°C and 60°C, and- incubation time ranging from 1 minute to 300 minutes; l) subsequently treating said Glucose Oxidase-treated protein slurry or isolate with Calcium lactate at a concentration ranging from 0.001 to 0.1 grams per gram of protein, followed by mixing for 5 to 30 minutes and pH neutralization; m) optionally treating said protein slurry or isolate with quillaia extract, wherein quillaia extract is present in an amount sufficient to enhance gelation and modulate texture; n) optionally combining said protein slurry or isolate with glucono delta lactone (GDL), in conjunction with or without said Calcium lactate and / or said quillaia extract, to further improve gelation properties; and o) wherein said resulting modified protein isolate exhibits enhanced gelation, structural integrity, and textural performance suitable for use in food applications selected from egg analogues, meat alternatives, dairy analogues, and baking ingredients.

2. The method as claimed in claim 1, wherein said milling is performed using one or more mills selected from hammer mills, pin mills, burr mills, or air classification mills, to achieve a particle size of less than 200 microns.

3. The method as claimed in claim 1, wherein said protein yield from the fractionation and extraction process exceeds 60% by weight of the starting plant material.

4. The method as claimed in claim 1, wherein said isoelectric precipitation step (step h) is omitted and protein is directly recovered from the supernatant to reduce processing time and cost.

5. The method as claimed in claim 1, wherein the combination of separation methods in step (i) includes ultrafiltration followed by nanofiltration with membrane pore sizes optimized based on the raw material used.

6. The method as claimed in claim 1, wherein the Glucose Oxidase treatment (step k) is conducted at a pH in the range of 4 to 8 and a temperature of of 25°C- 60°C for 1-300 minutes in the presence of 0.01 g glucose per unit of enzyme.

7. The method as claimed in claim 1 , wherein said Calcium lactate treatment (step l) is conducted after Glucose Oxidase treatment, with a predetermined dosage of protein and stirring for 15 minutes at room temperature.

8. The method as claimed in claim 1, wherein said quillaia extract is added in an amount ranging from 0.01% to 1% (w / w) relative to the protein content.

9. The method as claimed in claim 1, wherein said glucono delta lactone is added at a concentration of 0.05% to 0.5% (w / w) in combination with Calcium lactate and / or quillaia extract to synergistically enhance gelation.

10. The method as claimed in claim 1, wherein the resulting gelled protein product exhibits a gel strength of at least 200 g / cm2when measured using a texture analyzer under standard conditions.

11. A composition comprising a plant protein isolate prepared according to the method as claimed in claim 1, further characterized by: (a) protein content of at least 80% on a dry weight basis; (b) improved gelation profile when hydrated and heated, forming a thermally reversible or irreversible gel; (c) absence ofsynthetic crosslinkers, emulsifiers, or animal-derived additives; (d) clean-label formulation suitable for use in food systems requiring natural or minimally processed ingredients.

12. The composition as claimed in claim 11, wherein the protein is derived from mung bean and exhibits functional properties mimicking those of egg white proteins or whey proteins in baked or emulsified products.

13. A plant-based food product comprising the composition as claimed in claim 11, wherein said food product is selected from the group consisting of plantbased eggs, plant-based meat analogues, plant-based cheese, yogurt alternatives, and baked goods.

14. A method of improving the textural and gelation properties of a plant-based food product, the method comprising: a. incorporating a protein isolate or concentrate produced according to the method as claimed in claim 1 into a food formulation; b. subjecting the formulation to processing conditions suitable for gel formation, selected from thermal treatment, acidification, enzymatic treatment, or calcium-mediated crosslinking; and c. obtaining a final food product exhibiting enhanced textural attributes selected from improved firmness, cohesiveness, elasticity, or mouthfeel.

Images