DESC:FIELD OF THE INVENTION
[0001] The present disclosure pertains to a plant-based egg substitute. In particular, the present disclosure provides a process for the extraction of protein isolate from mung beans that can serve as a plant-based egg substitute. The present disclosure further relates to a composition comprising the mung bean protein isolate and formulations thereof.

BACKGROUND OF THE INVENTION
[0002] The 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] From ancient times, eggs have been consumed all over the world due to their nutritional value. Eggs have been known as a high protein source with a low caloric food source. Eggs are known for their functional attributes and therefore it is being used as emulsifier, gelling/thickening agent, foaming as well as water-binding agents in various food products.
[0004] Most commonly consumed eggs include chicken or hen eggs, also known as poultry eggs. Poultry eggs have high cholesterol and bacterial contamination. Moreover, they also contain disease-causing agents like Salmonella and Escherichia coli as well as bird viruses e.g., bird flu. Poultry eggs are also associated with animal cruelty and the negatives of animal farming. Industrial animal farming is also associated with the use of excessive antibiotics and hormones to enhance the growth and meat mass in animals. Hence, there has been a trend of people avoiding eggs due to personal and religious concerns as to exclude animal-based products and choosing a vegetarian or vegan diet.
[0005] In recent times, there has been a shift to food that is more sustainable and nutritious to improve the health of people as well as the planet. More and more people are becoming health conscious and are realizing the negative effects of animal-based foods on other humans, animals, surroundings, and the sustainability of the planet. In the world, out of 7.9 billion people in 2021, 79 million were vegans (Ethel Da Costa, “Veganism is the world’s new superpower”, blog, https://etheldacosta.com/, 28-Oct-2021). Increasing awareness about the disadvantages of animal-based products is driving the cultures toward a plant-based food philosophy. Plant-based foods are not only trending nowadays but are also becoming a need owing to health concerns about animal-based food.
[0006] Plant-based protein concentrates or isolates from plant sources are interesting ingredients for food applications, due to their functional properties and nutritional value. Therefore, there is a growing interest in plant-based proteins. Plant-based eggs are one such dietary replacements that provide superior or equivalent functionality eggs. Plant-based egg substitute offers customized nutritional and healthy food to address human dietary needs and at the same time reduce health risks such as cardiovascular disease. Plant-based egg substitute is used as a thickener in the bakery, confectionery, beverages, etc. However, it is not found suitable for cooking products like omelette/scrambled eggs. One such alternative available is plant-based egg liquid to overcome poultry eggs /shell-associated drawbacks. It comes in a liquid form in bottles, which can be readily used to cook omelettes and scramble eggs like a normal hen egg. However, it is not cost-effective from the consumer’s point of view. Also, the liquid egg alternative does not provide additional nutrients/micro-nutrients for supplementing the diet.
[0007] Hence, there exists a need to provide plant-based protein isolate that can serve as a plant-based egg substitute with one or more of the above-mentioned advantages and does not suffer from the shortcomings such as inadequate nutrients.
[0008] Plant seeds are a good source of protein. Mung bean, Vigna radiate (L.) Wilczek containing 17-26% protein is considered to be one such rich source of seed proteins. Especially, in developing countries, people are increasingly incorporating mung bean protein into their diets.
[0009] To meet such increasing demand for mung protein, there has been an attempt to extract protein from plants. Mostly plant protein extraction processes are generally developed according to final product specifications. Most methods involve protein extraction and purification by isoelectric precipitation of supernatant after solid/liquid separation from the slurry. Such methods of extraction and purification involve the use of acid/base chemicals, like sodium hydroxide, hydrochloric acid, and extreme alkaline and acidic conditions thereby adversely affecting the physical properties like protein denaturation, protein solubility, and related physicochemical properties. Also, digestibility can be impacted by these conditions, due to the loss of amino acid residues, like arginine, threonine, cysteine, lysine, and some other amino acid residues. Alkali-treated proteins can contain some unusual undesirable compounds, such as prolamins ornithine, lysinoalanine, ornithinoalanine, and lanthionine, as well as other D-amino acids. Further, unpleasant flavour compounds in beans/pulses are one of the main deterrents to acceptability of plant-based food products. Protein isolate derived from mung bean also suffers from undesirable beany earthy flavour and greenish bean colour, which remain the biggest challenges in food application such as bakery products, pasta, plant-based egg analogue, plant-based meat analogs and sausages as well as acceptability in users.
[00010] Accordingly, there still remains an unmet need to provide a process for extraction of plant-based protein isolate to serve as plant-based egg substitute obviating one or more aforementioned shortcomings of the existing processes and protein isolate especially for food application.
[00011] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

OBJECTS OF THE INVENTION
[00012] It is an object of the present disclosure to provide a process for extraction of protein isolate from mung bean that can serve as a plant-based egg substitute.
[00013] It is an object of the present disclosure to provide a process for extraction of protein isolate from mung bean that can help reduce or eliminate undesirable odour.
[00014] It is an object of the present disclosure to provide a process for extraction of protein isolate from mung bean that can help reduce or eliminate undesirable proteinaceous compounds and/or amino acids.
[00015] It is another object of the present disclosure to provide a composition comprising the mung bean protein isolate.
[00016] It is yet another object of the present disclosure to provide a plant-based egg scramble formulation comprising the composition containing the mung bean protein isolate.

SUMMARY
[00017] The present disclosure relates to a plant-based egg substitute.
[00018] In an aspect, the present disclosure provides a process for extraction of protein isolate from mung bean seeds that can serve as a plant-based egg substitute.
[00019] It is an aspect of the present disclosure that provides a process for extraction of protein isolate from mung beans that can help reduce or eliminate undesirable odour usually found in the mung bean protein isolate.
[00020] It is an aspect of the present disclosure that provides a process for extraction of mung bean protein isolate comprising steps of:
a) treating mung bean flour with ethanol;
b) removing the supernatant and drying treated mung bean flour;
c) extracting dried ethanol-treated mung bean with a salt solution for solubilizing protein;
d) separating insoluble and starch-rich compounds by filtering the solution;
e) centrifuging the filtered solution for separating a supernatant;
f) carrying out ultrafiltration by passing the supernatant solution through the filter membrane;
g) carrying out diafiltration by concentrating filtered supernatant through buffer exchange and passing through 10kda cassette; and
h) collecting concentrated mung bean protein isolate.
[00021] In another aspect, the present disclosure provides a composition comprising the mung bean protein isolate.
[00022] In an aspect, the present disclosure provides a composition comprising protein extract in the range from about 75 % to about 95 %, fat replacer in the range from about 5% to about 15%, emulsifier in the range from about 0.5% to about 5 %, hydrocolloid in the range from about 0.1% to about 0.5 %, and transglutaminase enzyme in the range from about 0.1% to about 2% w/v of the composition.
[00023] In yet another aspect, the present disclosure provides a plant-based egg scramble formulation comprising the composition containing the mung bean protein isolate.
[00024] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF DRAWINGS
[00025] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[00026] Figure 1 are photographic images showing: Figure 1(A) cross-linked base/acid extracted protein isolate obtained as per comparative method during cooking and Figure 1(B) after cooking.
[00027] Figure 2 are photographic images showing: Figure 2(A) the cross-linked water extracted protein isolate viscous mass obtained as per comparative method before cooking and Figure 2(B) after cooking.
[00028] Figure 3 are photographic images showing the cross-linked buffered extracted protein isolate obtained as per the comparative method after cooking.
[00029] Figure 4 is a photographic image showing the cross-linked NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure after cooking.
[00030] Figure 5 is a photographic image showing the cross-linked NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure after comparative dialysis followed by Ultrafiltration/Dia-filtration of mung bean protein isolate with 10 kDa Cassette.
[00031] Figure 6 is a photographic image showing the cross-linked NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure after comparative dialysis using a dialysis membrane (MWCO – 14 kDa) against water followed by Ultrafiltration/Dia-filtration of mung bean protein isolate with 10 kDa Cassette.
[00032] Figure 7 is a photographic image of cross-linked NaCl extracted protein isolate following membrane filtration and Ultrafiltration/Dia-filtration with 10 kDa Cassette using a buffer system in accordance with one of the exemplary embodiments of the present disclosure.
[00033] Figure 8 is a photographic image showing the composition comprising the cross-linked NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure after cooking.
[00034] Figure 9 are photographic images showing: the cross-linked composition comprising the NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure after cross-linking with transglutaminase enzyme at concentrations 0% (Figure 9(A)), 0.5% (Figure 9(B)) and 1% (Figure 9(C)).
[00035] Figure 10 are photographic images showing: the cross-linked composition comprising the NaCl extracted protein isolate obtained as per one of the exemplary embodiments of the present disclosure and individually different hydrocolloids Citrus fiber, Beta pectin, carboxymethyl cellulose, xanthan gum, high acyl gellan gum, low acyl gellan gum, high acyl gellan gum (85 % purity) and low acyl gellan gum (85 % purity) (Figures. 10(A)-(H)) respectively.

DETAILED DESCRIPTION OF THE INVENTION
[00036] The following is a detailed description of embodiments of the present disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[00037] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[00038] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[00039] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[00040] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. The term “about” whenever used in the context of and preceding numerical value, said numerical value can be construed as + 5 of that value. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[00041] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein.
[00042] All methods described herein can be performed in suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00043] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00044] Various terms are used herein. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[00045] The present disclosure relates to a plant-based egg substitute.
[00046] In certain embodiments, the present disclosure provides a process for extraction of protein isolate from mung bean that can serve as a plant-based egg substitute.
[00047] In an embodiment, the present disclosure provides a process for extraction of protein isolate from mung bean that can help reduce or eliminate undesirable odour usually found in the mung bean protein isolate.
[00048] In an embodiment, the present disclosure provides a process for extraction of mung bean protein isolate comprising steps of:
a) treating mung bean flour with ethanol;
b) removing supernatant and drying treated mung bean flour;
c) extracting dried ethanol-treated mung bean with the salt solution for solubilizing protein;
d) separating insoluble and starch-rich compounds by filtering the solution;
e) centrifuging the filtered solution for separating a supernatant;
f) carrying out ultrafiltration by passing the supernatant solution through filter membrane;
g) carrying out diafiltration for concentrating filtered supernatant by exchange through buffer system and passing through 10kda cassette; and
h) collecting concentrated mung bean protein isolate.
[00049] In one embodiment, treatment of mung bean flour with ethanol can be carried out by dispersing or suspending the mung bean flour in ethanol with the concentration range between about 70% to about 99.9% at a solid-to-liquid ratio in the range of about 1:4 to about 1:10 (w/V) and stirring at speed of about 50 - 400 rpm range with washing time between 4 to 20 hours to produce desirable mung bean flour.
[00050] In an embodiment, the ethanol-treated mung flour is separated from ethanol for example by filtration and drying.
[00051] In an embodiment, the wet ethanol-treated mung flour is dried in a hot oven at about 40 °C to about 80 °C for about 4 hrs to about 6 hrs or a period sufficient to provide dried mung bean flour. The dried ethanol-treated mung bean flour can be used for further processing or can be collected and stored in a clean container until required for further process.
[00052] In certain embodiments, the salt solution used for extraction of the ethanol-treated mung flour is sodium chloride (NaCl) solution.
[00053] In an embodiment, the sodium chloride solution used is of concentration 0.15 M to about 3 M.
[00054] In one embodiment, the ratio of ethanol-treated mung bean flour to NaCl solution is about 1:3 to about 1:10.
[00055] In one embodiment, the extraction of the ethanol-treated mung flour is carried out under stirring between about 100 rpm to 1000 rpm, preferably between 200 rpm to 800 rpm, preferably between 200 rpm to 600 rpm.
[00056] In one embodiment, the extraction of the wet ethanol-treated mung flour is carried out at a temperature of about 1 ºC to about 40 ºC, preferably at about 2 ºC to about 20 ºC, more preferably at about 2 ºC to about 10 ºC.
[00057] In one embodiment, the extraction of the ethanol-treated mung flour is carried out for a period of about 2 hours to 12 hours, preferably for about 4 hours to 8 hours.
[00058] In one embodiment, the separation of insoluble and starch rich compounds after extraction of the ethanol-treated mung flour is carried out by filtering the solution using suitable filtration means for example double-layered muslin cloth, or press filter or alternately using a decanter centrifuge.
[00059] In one embodiment, the centrifugation of the filtered solution of the extract for separating the supernatant is carried out at about 5000 rpm to about 20000 rpm, preferably at about 10000 rpm to 15000 rpm.
[00060] In an embodiment, the centrifugation is carried out at about 3 ºC to about 10 ºC.
[00061] In certain embodiments, to obtain increased protein yield, the residue from solid-liquid separation and centrifugation can be re-dispersed in NaCl solution as defined herein above. The dispersed solution can be mixed at between 400 rpm to 800 rpm for about 4 hours to 8 hours at about 2 ? to about 8 ?. Filtration and centrifugation steps are repeated as mentioned hereinabove. Supernatants from the two centrifugation steps are pooled to obtain the protein isolate solution.
[00062] In certain embodiment, the protein extract solution obtained is further subjected to steps of ultrafiltration and diafiltration to remove access salt in the extract.
[00063] Ultrafiltration is used for the production of mung bean protein concentrate with high protein content and diafiltration is used to obtain the final end product with increased purity.
[00064] In one embodiment, ultrafiltration is carried out by filtering the mung bean protein extract solution in step (f) through a membrane for example a 0.45-micron membrane.
[00065] The mung bean protein extract can be concentrated by diafiltration in step (g) using a buffer system solution and a tangential flow filtration (TFF) system.
[00066] In one embodiment, the buffer system comprises phosphate buffer and sodium chloride solutions.
[00067] In an embodiment, the phosphate buffer comprises one or more sodium phosphate monobasic monohydrate, Di-sodium hydrogen phosphate dihydrate, and Tetrasodium pyrophosphate.
[00068] In one embodiment, the phosphate buffer comprises sodium phosphate monobasic monohydrate about 0.5 M – 10 M, Di-sodium hydrogen phosphate dihydrate about 0.2 M – 2 M, and Tetrasodium pyrophosphate about 1 M -10 M.
[00069] In an embodiment, the sodium chloride used is of concentration 0.01 mM to about 400 mM.
[00070] In one embodiment, the buffer system comprises phosphate buffer comprising sodium phosphate monobasic monohydrate of about 0.5 to about 5%, Di-sodium hydrogen phosphate dihydrate of about 0.2% to about 2% and tetrasodium pyrophosphate of about 1 % to about 10%; and sodium chloride solution w/v.
[00071] In an embodiment, the buffer system has a pH of about 7 to about 8.
[00072] A polyether sulfone rectangular cassette used a total surface area of 110 cm2 and a molecular weight cut-off of 10 -100 kDa.
[00073] In a specific embodiment, the concentration and buffer exchange is performed at a transmembrane pressure (TMP) of about 2 bar and a feed flow rate of 25 - 500 mL.min-1. Measurements of water flux are carried out before and after ultrafiltration to quantify the formation of fouling on the membrane.
[00074] In one embodiment, the step of ultrafiltration is carried out in batch mode, where the initial volume is reduced to achieve a certain level of protein in the extract.
[00075] In another embodiment, ultrafiltration is carried out in discontinuous diafiltration mode. During diafiltration, the solution volume is kept constant by adding phosphate buffer, and simultaneously as filtrate is removed adding an equal amount buffer.
[00076] In an embodiment, protein-rich mung bean extract is concentrated to half of the initial volume by subjecting it to ultrafiltration followed by multiple diafiltration steps carried out with phosphate buffer. During the diafiltration cycle, phosphate buffer was added continuously at equivalent to the permeate volume to keep the constant volume of concentrate. During diafiltration, at every Dia-Volume (DV) run, the pH and conductivity are measured for the evaluation of buffer exchange. The process is run continuously and membrane and reservoirs are not cleaned between the concentration and purification steps of ultrafiltration and diafiltration. A chemical cleaning process is performed at the end of each experiment to restore the flux and retention characteristics of the membrane and prevent the growth of microorganisms in the system.
[00077] In one embodiment, the cleaning process comprises rinsing the membrane with distilled water, an alkaline cleaning, and the final cleaning with distilled water. The membrane is stored in the diluted alkaline solution. All experiments are conducted within the limits of pH, conductivity, and temperature tolerance of the membrane.
[00078] The steps of ultrafiltration and diafiltration help remove access amount of salt in the extract, also remove most of the low molecular weight compounds example, monosaccharides, disaccharides, protein impurities for example below 10 -100 kDa molecules, coloured compounds and increase the degree of protein purification.
[00079] In certain embodiments, the protein extract solution obtained after ultrafiltration and diafiltration is optionally subjected to treatment with activated carbon and pasteurization.
[00080] Optionally, the protein extract may be subjected to an activated carbon adsorption step to remove non-protein, off-flavour components, and additional fibrous solids from the protein extraction. The additional carbon adsorption step helps to obtain additional clarified and purified mung bean protein concentrate.
[00081] In certain embodiments, for activated carbon treatment, charcoal is used.
[00082] Charcoal is subjected to prior treatment for use as activated carbon. About 100 grams to about 300 grams of charcoal is mixed with about 3 kg of boiled water and poured through a filter. Again, the collected charcoal is mixed in about 3 kg of normal water, poured through a filter, and the charcoal is collected. The water-washed collected charcoal is dried in the hot air oven at 80 ?.
[00083] In one embodiment, about 1 gram to about 15 grams of treated charcoal is added to about 10 grams to about 200 grams of mung bean extract and incubated for about 30 minutes to about 120 minutes at about 2 ? to about 8 ?. The mixture is then filtered through double-layer muslin clothes to increase the purity and clarity of protein extract. The charcoal-treated extract is then filtered through a 0.45-micron membrane.
[00084] To avoid the possible spoilage of extract due to microbial contamination owing to many steps involved in the extraction and purification of mung bean protein, the mung bean protein extract may be subjected to pasteurization before use in food or storage.
[00085] In certain embodiment, the final protein extract resulting from salt extraction and membrane purification is pasteurized in a high temperature/short time pasteurization step to kill pathogenic bacteria that may occur during the extraction and purification process. In the particular embodiment, mung bean extract pasteurization is performed at about 45 ? to about 95 ? for about 1-3 seconds.
[00086] The extraction with a salt solution followed by ultrafiltration/diafiltration with a buffer system was surprisingly found to be the favourable method of extraction as compared to the alkaline extraction and acid precipitation method, water extraction method, and buffer extraction method as such or even when followed by ultrafiltration/diafiltration. Such unexpected effect of extraction with salt solution followed by ultrafiltration/diafiltration with buffer system can be determined with the help of cross-linking experimentation.
[00087] Cross-linking can be performed using a transglutaminase enzyme and calcium chloride solution as a cofactor for transglutaminase enzyme.
[00088] The microbial transglutaminase (MTGase) is a class of transferases enzyme used to modify the protein functional properties in food systems. Inter or intra-molecular cross-linking and polymerization are the main mechanisms of action of transglutaminase, resultant MTGase affects protein solubility, coagulation properties, gelation, viscosity, texture, emulsification, foaming, viscosity and water or oil holding capacity. Major reactions catalyzed by MTGase include acyl-transfer, cross-linking of lysine and glutamine amino acid residues, resultant a bridge formation called ?-(?-glutamyl) lysine bonds and deamination of protein molecules.
[00089] The protein isolate obtained in accordance with the process of the present disclosure can be characterized by subjecting the protein isolate to one or more physiochemical analysis such as composition, colour, volatile compound profile, SDS-PAGE, nitrogen solubility, water hydration and oil holding capacity, and emulsion stability by creaming.
[00090] Mung bean protein isolate obtained in accordance with the process of the present disclosure is found to be suitable for producing the egg-like texture upon crosslinking with transglutaminase enzyme.
[00091] In certain embodiments, there is provided a composition for cross-linking the mung bean protein isolate to form an egg analogue or scrambled egg-like structure.
[00092] In an embodiment, the present disclosure provides a composition comprising mung bean protein isolate from about 50% to about 95 %, oil from about 5% to about 16 %, emulsifier from about 0.1% to about 3 %, transglutaminase from about 0.5% to about 6 % and from about 0.5 M to about 1.5 M calcium chloride solution in the range of 25 µL to 400 µL.
[00093] In one embodiment, transglutaminase enzyme is added to the protein-rich solution at 0.1 – 2.0 % and blended with oil, emulsifier, and CaCl2 to make a homogenous mixture under medium to high shear mixing. The solution is heated to about 45 ºC - 75 ºC, preferably around 50 ºC (optimum reaction temperature for transglutaminase), and incubated for 60 minutes with stirring at about 50 rpm to about 150 rpm for homogenous reaction followed by cooking on a frying pan. During and after cooking of the cross-linked solution on the frying pan, the texture and structure of the solution and the end product is observed to resemble the scrambled egg.
[00094] The inventors of the present invention surprisingly found that the protein isolate obtained using a salt extraction method formed better cross-linking and desirable egg like structure and texture formation upon cooking with much less water separation during cooking and less cooking time requirement as compared to protein isolate prepared using alkaline extraction with acid precipitation, water extract or buffer extract.
[00095] Though the desirable scrambled egg like structure and texture is formed with the protein isolate obtained with the salt extraction method, the same possesses a high salt content.
[00096] It was unexpectedly found that further treatment of salt extracted protein isolate followed by ultrafiltration/diafiltration in presence of buffer system upon cross-linking provided much desirable scrambled egg like structure and texture with low salt content.
[00097] In another embodiment, there is provided an edible egg-free composition comprising a mung bean protein isolate obtained in accordance with the present disclosure.
[00098] In an embodiment, the egg-free composition comprises mung bean protein isolate, fat, emulsifier, hydrocolloid, enzyme, cofactor, and optionally excipients.
[00099] In one embodiment, the composition comprises about 70-90% mung bean protein isolate, oil about 5-15 %, hydrocolloid about 0.2-0.5 %, enzyme about 0.1% to about 2%, and cofactor about 0.01 – 0.1 %. The excipient can be about 0-25%.
[000100] The fat to be included in the composition can be selected from but is not limited to canola oil, sunflower oil, olive oil, coconut oil, soybean oil, palm oil, rape seed oil, mustard oil, flaxseed oil, cotton seed oil, and/or the combinations thereof to enhance the physical properties e.g. softness, leavening, coagulation and shine additionally. Also, as a replacement for poultry eggs in terms of fat, plant source fat or oil may be added as an alternative to the plant-based egg.
[000101] The emulsifier to be included in the composition can be selected from but not limited to soybean lecithin, sunflower lecithin, peanut lecithin, sesame lecithin, canola lecithin, or the combinations thereof.
[000102] The hydrocolloid to be included in the composition can be a suitable thickening agent or gelling agent selected from but not limited to high acyl gellan gum, low acyl gellan gum, iota-carrageenan, kappa-carrageenan, gum arabic, konjac gum, locust bean gum. guar gum, xanthan gum, citrus fibre, modified starch, cellulose, carboxymethyl cellulose, pectin, gelatin, agar, agarose, dextran or the combinations thereof.
[000103] The gellan gum can be high-acyl gellan gum or low-acyl gellan gum.
[000104] The enzyme to be included in the composition can be selected from but not limited to transglutaminase.
[000105] The cofactor to be included in the composition can be selected from but not limited to calcium chloride (CaCl2).
[000106] The excipients to be included in the composition can be selected from colouring agent, flavouring agent, preservative, antioxidant, pH modulators or the like.
[000107] The colouring agent used can be natural pigments, such as curcumin or carotenoids, typically to mimic the desirable yellowish colour of egg.
[000108] The flavouring agent can be those which can impart an egg like aroma and taste, or other desirable flavouring agent such as garlic powder, onion powder, sugars, salts, herbs, or spices.
[000109] The preservative that can be includes can be selected to prevent spoilage from bacteria, molds, fungi, or yeast; slow down or prevent changes in colour, flavour, or texture; delay rancidity and maintain freshness. The preservative can be selected from but not limited to ascorbic acid, citric acid, sodium benzoate, calcium propionate, calcium sorbate, tocopherol, or the like.
[000110] The pH modulator can be selected from but not limited to citrates, bicarbonates, or phosphates salt to control the pH.
[000111] The composition in accordance with the present disclosure can be prepared by blending mung bean protein isolate with oil, emulsifier and hydrocolloid in a defined amount to make a homogenous mixture under medium to high shear mixing. The mixture is then heated to temperatures of about 55 ?, followed by addition of transglutaminase enzyme and co-factor for the cross-linking. For the cross-linking of mung bean protein, the prepared mixture is incubated in thermal rotating incubator at about 55 ? for about 0.5-1.5 hours at 150-250 rpm. After cross-linking, the reaction mixture is quickly heated to > 70 ? for about 3-10 minutes to inactivate the transglutaminase enzyme.
[000112] The composition in accordance with the present disclosure upon cooking provides scrambled egg like structure and texture mimicking the scrambled egg prepared from poultry eggs.
[000113] Thus, the present disclosure fulfills an unmet need of an egg substitute in the form of mung bean protein isolate and composition comprising the same to provide plant-based egg analogue product, which is devoid of salt and mimics very closely the natural poultry eggs and such egg based products.
[000114] While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
Example 1
Ethanol Treatment of Mung bean flour
Materials:
Split mung bean pulses of brand Tata Sampann is procured from local shops grocery stores of Reliance Fresh, Pune, Maharashtra, India as per requirement. Ethanol was purchased from Changshu Hongsheng Fine Chemical Co., Ltd. China.
Method:
[000115] Mung bean flour dispersions were prepared by mixing the mung bean flour with absolute ethanol (99.9%) at a solid-to-liquid ratio of 1:4 (w/V) at 450 rpm overnight (15 ± 01 h) at an overhead stirrer at room temperature (25 °C). The ethanol-treated mung flour was obtained after single layer muslin clothes filtration, followed by hot oven drying of wet mung bean flour at 60 °C for 4 h. Dried ethanol-treated mung bean flour was collected in a clean container for further processing.
Example 2A
Comparative Extraction of protein isolate from mung bean
[000116] Extraction of mung bean protein from ethanol-treated mung bean flour was carried out as per the comparative conventional methods of alkaline extraction and acid precipitation, water extraction method and buffer extraction. Further, to determine the suitability of the protein isolate to be used as an egg substitute the cross-linking ability of the protein isolate was tested to form the egg like structure during and after cooking.
(A1). Comparative alkaline extraction and acid precipitation of mung bean protein isolate
(i). Extraction:
[000117] Ethanol-treated mung bean flour was dispersed in water using flour: water in ratios 1:3 to 1:5. The pH of the mixture was adjusted with 1M sodium hydroxide solution to alkaline pH between 9 -11 and the mixture was continuously stirred at 400 -600 rpm at 2-8 ºC for a period of 5 to 6 hours to maximize solubilization of the proteins. During this process, the alkaline pH was maintained throughout and the temperature was elevated to 4 ºC to 35 ºC to further enhance protein solubilization and extraction. The mixture was subsequently filtered to remove any insoluble material and the pH of the extract was adjusted to the isoelectric point by using 20 % citric acid solution to induce protein precipitation, followed by centrifugation to recover the protein isolate. The micro-Kjeldahl protein assay was used to measure the concentration of total protein in the mung bean extract sample, which was found to be ranging between 4.5 % to 5 % in the solution.
(ii). Cross-linking:
[000118] The protein extract solution (98.8 %), transglutaminase (1 %) and 1 molar of calcium chloride solution (0.2 %) as a cofactor for transglutaminase were blended and homogenized under medium to high shear mixing. The mix was heated at 50 ºC for 1 hour at 100 rpm, followed by cooking on a frying pan.
[000119] The cross-linked solution was cooked in a pan as a plant-based egg scramble. While during cooking lot of water separation was observed (Figure 1(A)). There was no egg-like texture or structure formed during and even after cooking, instead a thick jelly-like structure was formed (Figure 1(B)) and cooking time was also very high in the range of 3 to 6 minutes.
(A2). Comparative water extraction of mung bean protein isolate from ethanol-treated mung bean flour
(i). Extraction:
[000120] Ethanol-treated mung bean flour was dispersed in water using flour: water in ratios of 1:3 to 1:5. The mixture was continuously stirred at 400 -600 rpm at 2-8 ºC for a period of 1 hr to 10 hours to maximize solubilization of the proteins. The mixture was subsequently filtered to remove any insoluble material through double-layer muslin clothes followed by centrifugation and membrane filtration. The micro-Kjeldahl protein assay is used to measure the concentration of total protein in the mung bean extract sample, which was found to be between 3.2 % to 3.6 % in the solution.
(ii). Cross-linking:
[000121] To the solution of protein isolate (98%), transglutaminase enzyme (1 %) and calcium chloride solution (0.2 % of 1 M) were added and homogenized under medium to high shear mixing. The solution was heated to about 50 ºC (optimum reaction temperature for transglutaminase) and incubated for 60 minutes.
[000122] The cross-linked solution was cooked in a pan as plant-based egg scramble. After cross-linking with transglutaminase, many problems were observed with the water extract sample. The highly viscous solution (Figure 2(A)) was observed after cross-linking of the water extract sample during cooking, the egg-like structure and texture was formed but water separation was also observed. The texture of cooked cross-linked water extract sample was soft and watery (Figure 2(B)) and thus was not as desired.
(A3). Comparative buffer extraction of mung bean protein isolate from ethanol-treated mung bean flour
(i). Extraction:
[000123] Ethanol-treated mung bean flour was dispersed in buffer using flour: buffer in ratios 1:3 to 1:8. The composition of the buffer was the combination of tetrasodium pyrophosphate and sodium chloride, wherein tetrasodium pyrophosphate was in the range of 30 mM to 150 mM and sodium chloride was in the range of 0.15 M to 1.0 M. The mixture was continuously stirred at 400 -600 rpm at 2-8 ºC for period of 5 to 6 hours to maximize solubilization of the proteins. The mixture was subsequently filtered to remove any insoluble material through double-layer muslin clothes followed by centrifugation and membrane filtration. The micro-Kjeldahl protein assay is used to measure the concentration of total protein in the mung bean extract sample, which was found to be between 3.4 % to 3.8 % in the solution.
(ii). Cross-linking:
[000124] To the solution of protein isolate (98%), transglutaminase enzyme (1 %) and calcium chloride solution (0.2 % of 1 M) were added and homogenized under medium to high shear mixing. The solution was heated to about 50 ºC (optimum reaction temperature for transglutaminase) and incubated for 60 minutes. The cross-linked solution was cooked in a pan as plant-based egg scramble
[000125] After cross-linking the buffer extracted mung bean protein extract with enzyme, the solution obtained was slightly viscous, and after cooking, the texture was not as desired (Figure 3). Slightly water separation was also observed.
Conclusion:
[000126] All three methods that are (A1) comparative alkaline extraction and acid precipitation of mung bean protein isolate, (A2) comparative water extraction of mung bean protein isolate and (A3) comparative buffer extraction of mung bean protein isolate did not provide mung bean protein isolate that can be used as plant-based egg as a substitute to poultry egg to provide egg analogue product like scrambled egg upon cross-linking. Thus, none of these methods were found suitable to provide mung bean protein isolate as plant-based egg.
Example 2
Salt extraction of mung bean protein isolate from ethanol-treated mung bean flour as per an exemplary embodiment of the present disclosure
(i). Extraction:
[000127] Ethanol-treated mung bean flour 100 grams was dispersed in NaCl (0.5 M of 400 grams) solution. The extraction was performed under stirring at 600 rpm for 5 hours at 2-8 ?. Initially, double-layer muslin cloth was used to separate insoluble solid particles and starch-rich components from the slurry. To increase the purity of protein, the extract was centrifuged at 15,000 rpm for 30 minutes at 4 ?. The protein content was estimated by micro-Kjeldahl method was found to be 5.4 – 5.8 % in the solution.
(ii). Cross-linking:
[000128] To the solution of protein isolate (98%), transglutaminase enzyme (1 %) and calcium chloride solution (0.2 % of 1 M) were added and homogenized under medium to high shear mixing. The solution was heated to about 50 ºC (optimum reaction temperature for transglutaminase) and incubated for 60 minutes. The cross-linked solution was cooked in a pan as plant-based egg scramble.
[000129] After cooking the cross-linked salt extracted mung bean protein extract with enzyme, egg scramble-like coagulation was observed during cooking and egg scramble -like structure and texture were formed after cooking (Figure 4). There was less water separation during cooking and less cooking time was required as compared to acid/base extract, water extract or buffer extract. But after cooking, high salt content was experienced after taste. Hence, to make the salt extracted protein isolate suitable for food application, it was further processed.
(iii). Membrane filtration and ultrafiltration/diafiltration of mung bean protein
(iiia) Dialysis followed by Ultrafiltration/Dia-filtration of mung bean protein isolate with 10 kDa Cassette (Comparative method):
[000130] A dialysis experiment was performed after cross-linking. The salt extract of mung bean protein was dialyzed using a dialysis membrane (MWCO – 14 kDa) against water to remove excess salt from the solution. The majority of mung bean protein molecular weight is greater than 20 kDa. After dialysis, the cross-linking was carried out as per above step (ii). After cooking on the frying pan the egg-like structure and texture was not formed during cooking or after cooking. (Figure 5). Water separation was observed and cooking time was very high.
[000131] Diafiltration was carried out using laboratory scale Cogent® tangential flow filtration (TFF) system. 10 kDa hollow fiber was used to remove the excess salt from the extract and but after 4-5 DV the mung bean protein was precipitated in the solution. Further, when cross-linked with transglutaminase as per above step (ii), egg-like structure and texture were not formed (Figure 6).
(iiib). Membrane filtration and Ultrafiltration/Dia-filtration of mung bean protein isolate with 10 kDa Cassette using buffer system in accordance with the exemplary embodiment of the present disclosure:
[000132] Centrifuged mung bean protein extract obtained in step (i) was filtered through 0.8 µm membrane filter paper to increase the purity of mung bean extract (Protein content by micro Kjeldahl method – 5.79 %, pH -5.98, conductivity – 44.04 mS/cm, and NTU – 221).
[000133] Membrane-filtered mung bean extract was concentrated (2X) to increase the protein content (around 10 %) in the final extract by using a 10 kDa (MWCO) cassette of the area of 0.11 m2 on a Cogent µScale tangential flow filtration system.
[000134] A buffer exchange experiment was conducted without adding any acid and base to the buffer to remove access salt in the protein-rich extract for the application in food. 4 Dia- Volume (DV) of prepared phosphate buffer was used to remove excess salt in the particular experiment through the tangential flow filtration system.
[000135] The final retentate was collected and used as a plant protein source for plant-based egg scramble (Final extract conductivity – 17.48 mS/cm, pH – 7.13, NTU – 366 and protein content by micro Kjeldahl method – 9.2 ±0.3 %).
(iv). Cross-linking:
[000136] To the solution of protein isolate (98%), transglutaminase enzyme (1 %) and calcium chloride solution (0.2 % of 1 M) were added and homogenized under medium to high shear mixing. The solution was heated to about 50 ºC (optimum reaction temperature for transglutaminase) and incubated for 60 minutes. The cross-linked solution was cooked in a pan as plant-based egg scramble.
[000137] After cooking the cross-linked salt extracted mung bean protein extract with enzyme, egg scramble-like coagulation and no water separation were observed during cooking and egg scramble -like structure and texture were formed after cooking (Figure 7).
Example 3
Composition comprising protein isolate from mung bean
[000138] Following composition was followed for formulating plant-based egg analogue (scrambled egg):
[000139] Table 1: Composition comprising mung bean protein isolate:
Ingredients Quantity
Mung bean protein isolate (as per Example 2) 87.8 g
Canola Oil 9.5 g
Soya Lecithin Liquid 1.3 g
Gellan Gum (HA/LA) 0.25 g
Color (Curcumin) 12 mg
1 M CaCl2.2H2O Solution 200 µL
Microbial Transglutaminase 0.9 g
[000140] All the above ingredients were weighed and homogenized at 15,000 rpm for 4 minutes at room temperature to make a homogenous solution (especially oil in water emulsion). Homogenized solution was incubated at 55 ºC for 60 minutes at 180 rpm for cross-linking of the microbial transglutaminase enzyme of mung bean protein. The reaction mixture was quickly heated to 72 °C for 10 minutes to inactive the transglutaminase enzyme and also destroy the microorganisms that can spoil the end product.
[000141] With this composition, the final formulation performed very well. Egg-like structure and texture were formed during cooking and after cooking (Figure 8). Egg-like coagulation was observed and no water separation had been seen during cooking. Cooking time was also less as compared to all the above experiments.
Example 4
Compositions with and without transglutaminase enzyme
[000142] Compositions as per Example 3 were prepared except the concentration of transglutaminase enzyme, which varied from 0% to 1%. In absence of transglutaminase (0%) the egg like structure and texture were not formed (Figure 9(A)). Transglutaminase at both the concentrations that are 0.5% and 1% formed scrambled egg like structure and texture; however, 1% transglutaminase formed the most desirable scrambled egg like structure (Figures. 9(B)-9(C)).
Example 5
Compositions with different hydrocolloids
[000143] Compositions as per Example 3 were prepared with different hydrocolloids Citrus fiber, Beta pectin, carboxymethyl cellulose, xanthan gum, high acyl gellan gum, low acyl gellan gum, high acyl gellan gum (85 % purity) and low acyl gellan gum (85 % purity). All the hydrocolloids formed the scrambled egg like structure and texture (Figures. 10(A)-(H)).
[000144] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.
[000145] The present disclosure satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

ADVANTAGES OF THE INVENTION
[000146] The present disclosure provides a process for extraction of protein isolate from mung bean that can help reduce or eliminate undesirable odour.
[000147] The present disclosure provides a process for extraction of protein isolate from mung bean that can help reduce or eliminate undesirable proteinaceous compounds and/or amino acids.
[000148] The present disclosure provides a process for extraction of protein isolate from mung bean that can serve as a plant-based egg substitute.
[000149] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
,CLAIMS:1. A process for extraction of mung bean protein isolate comprising steps of:
a. treating mung bean flour with ethanol;
b. removing the supernatant and drying treated mung bean flour;
c. extracting dried ethanol-treated mung bean with a salt solution for solubilizing protein;
d. separating insoluble and starch-rich compounds by filtering the solution;
e. centrifuging the filtered solution for separating a supernatant;
f. carrying out ultrafiltration by passing the supernatant solution through the filter membrane;
g. carrying out diafiltration by concentrating filtered supernatant through buffer exchange and passing through 10kda cassette; and
h. collecting concentrated mung bean protein isolate.

2. The process as claimed in claim 1, wherein the ratio of mung bean flour to ethanol is in the range of about 1:4 to about 1:10 (w/V).

3. The process as claimed in claim 1, wherein the drying of ethanol-treated mung bean is carried out under stirring between about 100 rpm to 1000 rpm, preferably between 200 rpm to 800 rpm, preferably between 200 rpm to 600 rpm.

4. The process as claimed in claim 1, wherein the ethanol treated mung bean flour is dried at about 40°C to about 80°C.

5. The process as claimed in claim 1, wherein the ethanol treated mung bean flour is dried for about 4 hrs to about 6 hrs.
6. The process as claimed in claim 1, wherein the salt solution for solubilizing protein is sodium chloride (NaCl) solution.

7. The process as claimed in claim 6, wherein the sodium chloride solution is of concentration from about 0.15 M to about 3 M.

8. The process as claimed in claim 6, wherein the ratio of ethanol-treated mung bean flour to NaCl solution is about 1:3 to about 1:10.

9. The process as claimed in claim 1, wherein the extraction of the wet ethanol-treated mung flour is carried out at a temperature of about 1ºC to about 40°C, preferably at about 2°C to about 20°C, more preferably at about 2°C to about 10°C.

10. The process as claimed in claim 1, wherein the extraction of the ethanol-treated mung bean flour is carried out for a period of about 2 hours to 12 hours, preferably for about 4 hours to 8 hours.

11. The process as claimed in claim 1, wherein the centrifugation of the filtered solution of the extract for separating the supernatant is carried out at about 5000 rpm to about 20000 rpm, preferably at about 10000 rpm to 15000 rpm.

12. The process as claimed in claim 11, wherein the centrifugation is carried out at about 3°C to about 10°C.

13. The process as claimed in claim 1, wherein the diafiltration in step (g) uses a buffer system solution and a tangential flow filtration (TFF) system, and wherein the buffer system comprises phosphate buffer and sodium chloride solutions.

14. The process as claimed in claim 13, wherein the phosphate buffer comprises about 0.5 M – 10 M of sodium phosphate monobasic monohydrate, about 0.2 M – 2 M of Di-sodium hydrogen phosphate dihydrate, and about 1 M – 10 M of Tetrasodium pyrophosphate.

15. The process as claimed in claim 13, wherein the buffer system has a pH of about 7 to about 8.

16. The process as claimed in claim 1, wherein the buffer exchange is performed at a transmembrane pressure (TMP) of about 2 bar and a feed flow rate of 25 – 500 mL.min-1.

17. The process as claimed in claim 1, further comprises treating with activated carbon to remove non-protein, off-flavour components, and additional fibrous solids from the protein isolate.

18. The process as claimed in claim 17, wherein about 10 grams to about 200 grams of mung bean isolate is treated with about 1 gram to about 15 grams of treated charcoal and incubated for about 30 minutes to about 120 minutes at about 2? to about 8?.

19. The process as claimed in claim 1, further comprises pasteurizing to remove pathogenic bacteria that may occur during the extraction and purification process.

20. The process as claimed in claim 17, wherein the mung bean isolate is pasteurized at about 45 ? to about 95 ? for about 1-3 seconds.
21. An egg-free composition comprising mung bean protein isolate from about 70 to 90%, oil from about 5 to 15 %, hydrocolloid from about 0.2 to 0.5 %, enzyme about 0.1% to about 2%, and cofactor from about 0.01 to 0.1 %.

22. A composition comprising mung bean protein isolate from about 50% to about 95%, oil from about 5% to about 16%, emulsifier from about 0.1% to about 3%, transglutaminase from about 0.5% to about 6 % and from about 0.5M to about 1.5M calcium chloride solution in the range of 25 µL to 400 µL.

23. A process for preparing the composition comprising mung bean protein isolate as claimed in claim 22 comprises the steps of
a) adding transglutaminase enzyme to the mung bean protein isolate at 0.1 – 2.0 %;
b) blending with the mixture from step a) oil, emulsifier, and CaCl2 to make a homogenous mixture;
c) heating homogenous mixture of step b) to about 45ºC – 75ºC;
d) incubating the mixture from step c) for 60 minutes with stirring at about 50 rpm to about 150 rpm; and
e) cooking on a frying pan to obtain the end product resembling scrambled egg.