Description:FIELD OF INVENTION
The present invention relates to the field of isolation and purification of ribulose 1,5-bisphosphate carboxylase / oxygenase (RuBisCO). The present invention more particularly relates to a method for isolating and purifying a RuBisCO protein from leftover plant material.
BACKGROUND
The growth of overall population and shifts in demographic patterns have resulted in a sharp need for a variety of food choices, particularly focusing on plant-derived proteins. The primary reason for this phenomenon can be attributed to the negative effects on the ecosystem associated with the meat industry, such as increased emissions of greenhouse gases and the substantial consumption of water and land resources (Refer 1, Henchion et.al).
The increasing demand for plant-derived proteins has led to the rapid development of intensive farming methods using significant resources and modern technologies in recent years. In addition to the growing reliance on plant-based proteins, there has been a corresponding increase in the number of residual products generated, particularly the leftover plant residues, which could contribute to landfill overflow and environmental pollution. Therefore, there is a need for a viable solution (Refer 2, Raza et.al).
Ribulose bisphosphate carboxylase/oxygenase (RuBisCO) is a plant protein of significant interest due to its nutritional and functional properties. It is a complete protein ideal for human consumption and has the potential to serve as a valuable food additive due to its biochemical composition, organoleptic characteristics, and physical features. Despite its potential health benefits, the lack of a streamlined extraction/isolation, and purification processes at an industrial scale poses significant challenges to its commercial significance (Refer 3, Nawaz et.al).
The contamination of plant materials with harmful substances, such as chemicals, heavy metals like cadmium, mercury, lead, and arsenic, as well as pollutants from the environment such as hormones and pesticides, presents a significant challenge in obtaining purified RuBisCO. It is imperative to meticulously design and implement extraction and purification methods for this crucial plant enzyme to guarantee the absence of any harmful contaminants in the final product. This is an arduous task (Refer 4, Quintieri et.al). Therefore, acquiring RuBisCO protein isolate that is adequately purified for consumption can present a considerable and complex challenge.
Many of the currently known purification methods lead to RuBisCO proteins isolates that having a greenish tint due to high levels of chlorophyll, which may not be suitable for all types of food and beverages. Applying heat to partially solidify the pigmented substance can aid in its removal in subsequent processing stages. However, the use of heat may lead to denaturation or aggregation of the proteins, resulting in the loss of essential nutrients, reduced solubility, or modifications in specific nutrients. It is essential to devise methods with suitable heat treatment that effectively manage these variables to achieve RuBisCO proteins without green tint. Moreover, many isolation methods for RuBisCO lead to proteins contaminated with heavy metals, anti-nutritional elements and toxins.
Given the aforementioned limitations, the scope of the present invention relies on a carefully constructed isolation and purification method for extracting RuBisCO protein isolate from residual plant leaves.
SUMMARY
The present invention describes a sustainable method of isolating highly pure RuBisCO protein from plant leaves the method comprising the steps of:
a. homogenizing cleaned plant leaves by using water with pH adjusted to 8.0-8.3; with leaf: water ratio as 1: 2 (w:v) to make a leaf slurry;
b. sieving the leaf slurry obtained in step (a) and collecting the filtrate and the fibre left in the sieve;
c. centrifuging the filtrate from step (b) and collecting supernatant-1 and pellet-1;
d. subjecting the supernatant-1 to thermal coagulation at 42-47°C;
e. centrifuging the thermal coagulated liquid from step (d) and collecting both supernatant-2 and pellet-2, wherein pellet-2 comprises 60-74% pure RuBisCO protein;
f. subjecting the supernatant-2 from step (e) to acid precipitation at pH 4.5; and
g. centrifuging the acid precipitated supernatant-2 from step (f) to obtain pellet-3, wherein pellet-3 comprises 75-80% pure RuBisCO protein.
In one embodiment, the plant leaves selected are from spinach, sweet potato, broccoli, cauliflower, cabbage, banana, or papaya plants.
In one embodiment, the homogenization of the plant leaves in step (a) of the mentioned method is done by mechanical means.
In one embodiment, the sieving in step (b) of the mentioned method is done with a sieve with 400 mesh size.
In one embodiment, the centrifugation in step (c) of the mentioned method is to collect the supernatant-1 is done at 12000-16,000g for 15-20 mins at 2-8°C.
In one embodiment, the thermal coagulation of the supernatant-1 in step (d) of the mentioned method is done by incubating it at 42-47°C for 10 mins and cooling down to room temperature (RT).
In one embodiment, the centrifugation of the thermal coagulated liquid in step (e) of the mentioned method is done at 12000-16000g for 15-20mins at 2-8°C.
In one embodiment, the acid precipitation of the supernatant-2 in step (f) of the mentioned method is done with 1N HCl.
In one embodiment, the acid precipitation of the supernatant-2 in step (f) of the mentioned method is done for 8-12 hours.
In one embodiment, the centrifugation of the acid precipitated supernatant-2 of the mentioned method is centrifuged at 12000-16000g for 15-20mins at 4°C.
In one embodiment, the pellet-2 and the pellet-3 of the mentioned method comprise high purity RuBisCO protein.
In one embodiment, the purified RuBisCO protein in pellet-2 and pellet-3 of the mentioned method is free of toxins, anti-nutritional substances and heavy metals.
In one embodiment, the pellet-1 collected after step (c) of the mentioned method comprises 30-35% pure RuBisCO protein.
In one embodiment, the fibre left in the sieve after homogenization and filtration of the plant leaf slurry in step (b) of the mentioned method is also collected and comprises 15-18% pure RuBisCO protein.
BRIEF DESCRIPTION OF FIGURES:
Figure 1 (a- i) shows different stages of RuBisCO protein extraction from (a) Broccoli leaves, (b) pooled homogenate, (c) Supernantant-1, (d) Fiber, (e) pellet-1, (f) thermal coagulation, (g) pellet-2, (h) Supernatant-3 and (i) Pellet-3.
Figure 2a & 2b: Lyophilised RuBisCO protein powders from Broccoli leaves. (a) Off-white protein powder, (b) Green protein powder
Figure 3 Coomassie Blue stained SDS-PAGE showing the protein extraction from broccoli leaves at various stages.
1. BSA - Bovine serum albumin factor V standard/M= Marker
2. HL - Homogenized liquid
3. Pool - Pooled green liquid after 2 step homogenization and sieving
4. CFS - Centrifuged supernatant-1
5. CFP - Centrifuged pellet-1
6. CFPS - Centrifuged supernatant-1 where pH was adjusted to 8.3
7. TS - Supernatant-2 obtained after thermal treatment
8. TP - Pellet-2 obtained after thermal treatment
9. AS - Supernatant-3 obtained after acid precipitation
10. AP - Pellet-3 obtained after acid precipitation
Figure 4 shows Coomassie Blue stained SDS-PAGE showing the protein extraction from cauliflower leaves at various stages.
1. BSA - Bovine serum albumin factor V standard
2. HL - Homogenized liquid
3. Pool - Pooled green liquid after 2 step homogenization and sieving
4. CFS - Centrifuged supernatant-1
5. CFP - Centrifuged pellet-1
6. TS - Supernatant-2 obtained after thermal treatment
7. TP - Pellet-2 obtained after thermal treatment
8. AS - Supernatant-3 obtained after acid precipitation
9. AP - Pellet-3 obtained after acid precipitation

DETAILED DESCRIPTION OF THE INVENTION
This section is intended to provide an explanation and description of various possible embodiments of the present invention. The embodiments used herein, and the various features and advantageous details thereof are explained more fully with reference to non-limiting embodiments illustrated in the accompanying drawing/s and detailed in the following description. The examples used herein are intended only to facilitate an understanding of ways in which the embodiments may be practiced and to enable the person skilled in the art to practice the embodiments used herein. Also, the examples/embodiments described herein should not be construed as limiting the scope of the embodiments herein.
For convenience, the meaning of certain terms and phrases used in here, are provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
Unless specifically stated otherwise, a process or method comprising multiple steps may include additional steps at the beginning or end of the method or may include additional intervening steps. Also, the steps may be combined, excluded, or performed in an alternate order, as appropriate.
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
Many of the currently known purification methods lead to RuBisCO proteins isolates that have a greenish tint due to high levels of chlorophyll, which may not be suitable for all types of food and beverages. Applying heat to partially solidify the pigmented substance can aid in its removal in subsequent processing stages. However, the use of heat may lead to denaturation or aggregation of the proteins, resulting in the loss of essential nutrients, reduced solubility, or modifications in specific nutrients. It is essential to devise methods with suitable heat treatment that effectively manage these variables to achieve RuBisCO protein isolates without green tint. Moreover, many isolation methods for RuBisCO lead to proteins contaminated with heavy metals, anti-nutritional elements and toxins.
Also, many protein purification techniques for RuBisCO rely on thermal treatments at temperatures above 70 °C, which leads to loss of many protein characteristics such as foaming, gelling, which in turn hinders use of this protein for downstream processes.
Given the aforementioned limitations, the scope of the present invention relies on a carefully constructed isolation and purification method for extracting RuBisCO protein from residual plant materials, such as leaves.
RuBisCO protein
RuBisCO, also known as Ribulose-1,5-bisphosphate carboxylase/oxygenase, has garnered considerable attention in the academic as well research community owing to its nutritional and functional attributes. This plant-based protein is currently acknowledged as a valuable protein source for individuals following vegetarian and vegan diets. The potential utilization of RuBisCO as a food ingredient arises from the inadequacies of commercially available proteins, which often fall short in meeting essential nutritional needs, posing allergenic risks, lacking crucial amino acids, or exhibiting unpalatable flavors. By enhancing the production and purification processes of RuBisCO, a non-allergenic and easily digestible protein present in plant tissues, it is plausible to tackle global challenges associated with protein deficiency. Despite possessing the requisite characteristics for a protein suitable for human consumption or animal feed, the impediment to its widespread commercialization persists due to the complexity of isolating and purifying RuBisCO in a scalable manner without retaining its green pigment.
DEFINITIONS
The term “heavy metals” as used herein, refers to lead (Pb), cadmium (Cd), copper (Cu), arsenic (As), mercury (Hg) and zinc (Zn).
The term “plant pollutants” as used herein, refers to sulphur dioxide, fluorine, peroxyacetyl nitrate, and derivatives.
The term “anti-nutritional factors/ elements” or “anti-nutrients” refer to compounds which reduce the nutrient utilization and/or food intake of plants or plant products used as human foods. Anti-nutritional factors reduce the beneficial effects of plant foods, by inhibiting absorption or having some deleterious effects reducing optimum nutrition. Such anti-nutritional compounds, are mostly, but not always with food of plant origin. Examples of “anti-nutritional factors” include, but are not limited to tannins, saponins, lectins, oxalate, protease inhibitors, cyanogenic glycosides, and alkaloids.
The term “thermal coagulation of proteins refers to the process by which proteins undergo irreversible structural changes and aggregate upon exposure to heat (often above 37°C & rarely even going upto 70-80°C), leading to their precipitation or gelation. This phenomenon occurs due to the disruption of weak non-covalent bonds within the protein's tertiary and quaternary structures, resulting in loss of solubility and functional activity.
The term “acid precipitation” of proteins involves the process of selectively precipitating proteins from a solution by adjusting the pH to a point where the protein's net charge becomes neutral or positive, causing it to aggregate and precipitate out of solution. This method takes advantage of the protein's isoelectric point (pI), which for most protein ranges between 4-6 where it is least soluble, effectively separating it from other components
“Foam ability (ability to form foam)” and “foam stability” are important functional properties of proteins essential in many food formulations.
The property of proteins to form stable foams is important in the production of a variety of foods. The unfolding of the protein structure and the diffusion at the air–water interface are related to the foaming capacity (FC) and the viscoelastic film formed around the bubbles is associated with the foam stability (FS).
Foam is usually defined as a two-phase system consisting of air cells separated by a thin continuous liquid layer called the lamellar phase. Food foams are usually very complex systems, including a mixture of gases, liquids, solids, and surfactants. Body and smoothness of food foams are also important properties and the proteins in the foods can affect these too.
As used herein the term “Foam stability” is measured as the time required to lose either 50% of the liquid or 50% of the volume from the foam.
Emulsion stability as used herein is defined stability of the emulsion layer formed at oil-water interface in foods, which is stabilised by the proteins or peptides adsorbed at the interface. Oil-in-water emulsions exist in foods that contain oil or fat. The proteins form a protective steric barrier around the oil droplets. Emulsion stability measured by the kinetics of the change in the size distribution or the spatial arrangement of droplets over a time-period.
EMBODIMENTS
Ensuring the purity of RuBisCO, a crucial plant protein, is a challenging task due to the potential contamination of plant materials with various harmful substances. It is essential to carefully plan and execute isolation and purification processes to eliminate any contaminants and obtain a safe product. This process can be difficult and complex, making it important to obtain precisely purified RuBisCO for consumption. Various purification methods can result in RuBisCO proteins acquiring a greenish hue from high chlorophyll levels, which may not be ideal for all food and beverage applications. Applying heat to partially solidify the pigmented substance can assist in its removal during processing. However, the use of heat can potentially cause denaturation or aggregation of the proteins, leading to the loss of nutrients, reduced solubility, or alterations in specific nutrients. It is important to develop methods that incorporate appropriate heat treatment to effectively address these issues and produce RuBisCO proteins without a green tint.
Considering the aforementioned constraints, the focus of the current invention hinges on a meticulously designed method for isolating and purifying RuBisCO protein from leftover plant materials.
Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the embodiments herein with modifications.
The present invention describes a sustainable method of isolating highly pure RuBisCO protein from plant leaves the method comprising the steps of:
a) homogenizing cleaned plant leaves by using water with pH adjusted to 8.0-8.3; with leaf: water ratio as 1: 2 (w:v) to make a leaf slurry;
b) sieving the leaf slurry obtained in step (a) and collecting the filtrate and the fibre left in the sieve;
c) centrifuging the filtrate from step (b) and collecting supernatant-1 and pellet-1;
d) subjecting the supernatant-1 to thermal coagulation at 42-47°C;
e) centrifuging the thermal coagulated liquid from step (d) and collecting both supernatant-2 and pellet-2, wherein pellet-2 comprises 60-74% pure RuBisCO protein;
f) subjecting the supernatant-2 from step (e) to acid precipitation at pH 4.5; and
g) centrifuging the acid precipitated supernatant-2 from step (f) to obtain pellet-3, wherein pellet-3 comprises 75-80% pure RuBisCO protein.
In one embodiment, the plant leaves selected are from spinach, sweet potato, broccoli, cauliflower, cabbage, banana, or papaya plants.
In one embodiment, the homogenization of the plant leaves in step (a) of the mentioned method is done by mechanical means.
In one embodiment, the sieving in step (b) of the mentioned method is done with a sieve with 400 mesh size.
In one embodiment, the centrifugation in step (e) of the mentioned method is to collect the supernatant-1 is done at 12000-16,000g for 15-20 mins at 2-8°C.
In one embodiment, the thermal coagulation of the supernatant-1 in step (d) of the mentioned method is done by incubating it at 42-47°C for 10 mins and cooling down to room temperature (RT).
In one embodiment, the centrifugation of the thermal coagulated liquid in step (e) of the mentioned method is done at 12000-16000g for 15-20mins at 2-8°C.
In one embodiment, the acid precipitation of the supernatant-2 in step (f) of the mentioned method is done with 1N HCl.
In one embodiment, the acid precipitation of the supernatant-2 in step (f) of the mentioned method is done for 8-12 hours.
In one embodiment, the centrifugation of the acid precipitated supernatant-2 of the mentioned method is centrifuged at 12000-16000g for 15-20mins rpm at 4°C.
In one embodiment, the pellet-2 and the pellet-3 of the mentioned method comprise high purity RuBisCO protein.
In one embodiment, the purified RuBisCO protein in pellet-2 and pellet-3 of the mentioned method is free of toxins, anti-nutritional substances and heavy metals.
In one embodiment, the pellet-1 collected after step (c) of the mentioned method comprises 30-35% pure RuBisCO protein.
In one embodiment, the fibre left in the sieve after homogenization and filtration of the plant leaf slurry in step (b) of the mentioned method is also collected and comprises 15-18% pure RuBisCO protein.
In one embodiment, the purified RuBisCO protein obtained from the mentioned method is devoid of phytochemicals or anti-nutritional factors such as Glucosinolates, Isothiocyanates, Lectins, Oxalates, and their derivatives and the like.
In one embodiment, the purified RuBisCO protein obtained from the mentioned method is devoid of anti-nutritional substances.

In one embodiment, the purified RuBisCO protein obtained from the mentioned method is devoid of heavy metals.
In one embodiment, the purified RuBisCO protein obtained from the mentioned method is devoid of plant pollutants.
In one embodiment, the plant leaves selected are from Brassicaceae or Cruciferae, Caricaceae, Amaranthaceae, Musaceae, or Convolvulaceae.

In one embodiment, the method mentioned allows for the purification of RuBisCO protein with essential nutrients. This is achieved through a designed method that involves heating supernatant-1 at 42-47°C for 10 minutes and then cooling it to room temperature for the thermal coagulation step. The thermally coagulated liquid is then centrifuged at 12000-16000g for 15-20 minutes at 2-8°C in another step of the process.
In one embodiment, the method disclosed herein allows for the isolation and purification of Rubisco protein with essential amino acids.
In one embodiment, the purified Rubisco protein obtained from the mentioned method is tasteless, odourless, off-white or colourless, and a neutral protein.
In one embodiment, the purified Rubisco protein obtained from the mentioned method is suitable for food and beverage industries and the like.
EXAMPLES
Example 1: Extraction of off-white RuBisCO protein from Broccoli leaves
1.1 Leaf preparation process for RuBisCO protein isolation:
? Freshly harvested Broccoli leaves (Fig. 1a) collected for extraction of off-white RuBisCO protein isolate.
? Deveining process: Remove hardy mid-rib from leaves and obtained leaf blades were subjected to further processing.
? Subsequently, the leaves underwent surface sterilization using Tween 20 (5%) to eliminate microbial and other contaminants, followed by two thorough washes under running tap water to remove dirt and dust particles and also to ensure removal of tween 20.
? Leaves were finally rinsed with double distilled water (DDW) to remove any residual impurities that could affect protein extraction.
1.2 Homogenization of leaves:
? The cleaned leaves were cut into small pieces to facilitate homogenization. Double distilled water was used for homogenizing the leaves. Check and maintain the pH of the water at 8-8.5 using 1M NaOH/1N HCl.
? Mechanically the leaves were homogenized using a blender by adding the pH-regulated DDW. Leaf to water ratio of 1:0.75 to 1:2 was maintained for different leaves used.
? For the first set of homogenization half the volume of water was used which ensured the extraction of soluble protein into the homogenate while maintaining its stability and integrity. In order to guarantee optimal cytoplasmic protein recovery, the homogenization process's speed and duration of time were carefully chosen for cell wall breakage. The optimal duration for homogenization was found to be 3–4 minutes, divided into an equal number of spin cycles 45-seconds each.
? The initial homogenate obtained was sieved using a fine 400-mesh size to separate the green juice. The pulp obtained was again subjected to a second cycle of homogenization using rest of pH adjusted water to make sure that the cell wall gets ruptured completely and results in maximum protein recovery into homogenate.
? The obtained homogenate was again sieved using the same mesh and the green juice obtained in first and second homogenization were pooled together for further processing (Fig. 1b). The fiber separated was oven dried at 45°C overnight to ensure it free from moisture and further testing was done (Fig. 1d).
? The combined green juice was centrifuged using parameters between 12000-16000g for 15 to 20 minutes at a temperature between 2-8°C to guarantee the total separation of the insoluble component from the soluble protein fraction.
? The Supernatant-1 (light brown colour juice) was collected and utilized for further processing after centrifugation. The pellet-1 (green pellet) (Fig. 1e) was collected, freeze dried and stored as a green protein fraction.
1.3 Thermal coagulation of the brown juice:
• The collected supernatant-1 was subjected to thermal coagulation or regulated thermal treatment at 42-47°C (Table 1). This process involves heating the juice at 45°C while continuously stirring until the temperature is uniformly distributed throughout the liquid for 10 minutes. This heating step is crucial as it facilitates the coagulation of green proteins (Fig. 1f) present in the juice.
• Once the heating phase was completed, the hot brown juice was allowed to cool down to room temperature. This step ensured the coagulation of the green protein effectively.
• Subsequently, the cooled brown juice was subjected to another round of centrifugation at 12000-16000 g for 15-20 minutes at 2-8°C. This centrifugation separated the mixture into two components: the pellet-2 (Fig. 1g) and the supernatant-2. The pellet obtained from this centrifugation step is referred to as green protein isolate which is light green in colour. The green protein isolate can be used as a nutritionally active binding agent in plant-based meat products and similar applications. The supernatant-2 was brown in colour and used for further processing.
1.4 Acid precipitation of RuBisCO protein:
? The supernatant-2 obtained after centrifugation was then processed through isoelectric precipitation by adjusting the pH of the liquid to the 4.5 with 1N HCl. Once the target pH is achieved, the solution is left to stand for 8-12 hours at a temperature between 2-8°C. This precipitation step is followed by another round of centrifugation at 12000-16000g for 15-20 minutes at 2-8°C. This step resulted in pellet-3 and supernatant-3 (Fig. 1h & i). The obtained acid precipitation pellet was off-white in colour which is referred as off-white protein isolate. The pellet was subjected to freeze drying, while the supernatant was discarded at this stage.
? Subsequently, each pellet - the acid pellet, thermal pellet, and CF pellet - undergoes separate freeze-drying processes to ensure complete removal of moisture and obtain fine powders. These powders are then carefully packaged under moisture-free conditions (Fig. 2a & b).
Table 1: Comparative analysis of various coagulation & precipitation parameters w.r.t protein isolate recovery.
Protocol No. of batches Colour of the pellet Avg wt of pellet (pellet-2 and pellet-3)(g/Kg of leaf weight) (yield)
Thermal coagulation 55°C CF (Centrifuged liquid) pH adjusted to 8.3 3 Light green 26.93
CF pH not adjusted 2 25.32g
45°C CF pH adjusted to 8.3 3 Light green 19.27
CF pH not adjusted 3 9.22
Acid precipitation 55°C CF pH adjusted to 8.3 3 Off white/ cream 21.13
CF pH not adjusted 2 13.9g
45°C CF pH adjusted to 8.3 3 29.95
CF pH not adjusted 3 27.02
Combined thermal and acid precipitation 50°C 2 Light green
81.86
60°C 2 79.32
70°C 2 85.85

? Initial experiments were carried out at different temperatures 50°C, 60°C and 70°C and once the temperature is reached the pH of centrifuged liquid was adjusted to 4.5 which resulted in green colour pellet.
? The separation of steps i.e. thermal treatment and acid treatment at 45°C resulted in maximum yield of off-white protein isolate.
Example 2: Analysis and Evaluation of the extracted RuBisCO:
? The resulting protein powder was analyzed by several experiments, including SDS-PAGE analysis and evaluation of other functional parameters such as gelation, foaming, and emulsification. The SDS-PAGE analysis resulted in presence of RuBisCO bands of ~55 KDa and ~15 KDa (Fig. 3 & 4).
? This protocol resulted in two distinct protein products: a green protein isolate (yield was found to be 19.27 g/Kg WW of leaf / 3-4g/kg DW of leaf with 60-70% protein purity) and an off-white protein pellet (yield was observed to be 29.95 g/Kg 7-7.5 g/kg DW with 75-80% protein purity), both suitable for large-scale production.
? Two intermediate products were also obtained during the process i.e. green fiber (220g/kg WW/25g/kg DW with 15% protein content) and green centrifuge pellet (135 g/kg WW/ 35 g/kg DW @30-35% protein content).
? The resulting leaf protein isolate exhibits high solubility, pleasing taste, and has no leafy odor, making it an ideal ingredient for various food formulations.
2.1 Functional properties of the green and off-white protein isolates:
? The protein isolate was subjected to various functional attributes checks. The green protein pellet showed foam expansion of 41.6% and foam stability of around 76.4%. The off-white colored acid precipitation pellet showed the same foam expansion of 41.6% whereas foam stability of 100% (Table 2). Foaming test was done as given in Ref 5 (Giardina, C., et al).
? Both the protein isolates showed excellent emulsion stability of 100% and 98% respectively. as well as almost 70% binding capacity (Table 3). Emulsion test was done as given in Ref 6 Mcclements, D. J. et al.
Table 2: Foaming Capacity and Stability Test
Protein Initial sample volume (Vliquid) Initial foam volume (V0) Foam volume after 2h (Vt) Foam expansion % Foam stability%
Green protein isolate 6 ml 8.5 6.5 ml 41.6% 76.4%
Off-white protein isolate 6 ml 8.5 8.5 ml 41.6% 100 %

Table 3: Emulsion Stability Test
Protein Volume of aqueous phase before emulsification (VB) Volume of aqueous layer after emulsification (VA) Emulsion stability%
Green protein isolate 5 ml 1.1 ml 78 %
Off-white protein isolate 5 ml 1.25 ml 75%

Example 3: Quality Analysis :
? Proximate Analysis: Proximate analysis is a technique to measure the chemical properties of a compound based on four particular elements: moisture content, fixed carbon, volatile matter and ash content (Ref 7 Leela et al. 2016; Ref 9 Sharuddin, S. D. A et al ). Various components such as crude proteins, fats, fibers, carbohydrates, ash, and moisture content were quantified using validated methods [modified AOAC (Association of Official Agricultural Chemists) methods].
? Mineral Analysis: Elemental analysis of these isolates through EDAX proved that there were no traces of harmful heavy metals. LC/MS studies proved that our protocol eliminates harmful toxin and other anti-nutritional components from the final products. Mineral analysis was done as given in Ref 8 : Rao, R. et al.
Applications:
? The off-white protein isolate can be used as an active ingredient in health drinks, fortified protein bars etc.
? The green protein isolate can be used as a nutritionally active binding agent in plant-based meat products and similar applications.
? The fiber by-product recovered can be used as cattle feed/fodder and animal feed with high nutritionals value along with the other in-process bi-product which is the centrifuged green pellet also boasts of decent nutritional parameters.
This protocol is a zero-waste, sustainable protocol; each product, by-product has distinct use cases in the plant-based food industry.

References
1. Henchion, M., Hayes, M., Mullen, A.M., Fenelon, M., Tiwari, B. (2017) Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium. Foods. 6(7):53.
2. Raza, M.H., Abid, M., Faisal, M., Yan, T., Akhtar, S., Adnan, K.M.M. (2022) Environmental and Health Impacts of Crop Residue Burning: Scope of Sustainable Crop Residue Management Practices. Int J Environ Res Public Health. 19(8):4753.
3. Nawaz, M.A., Kasote, D.M., Ullah N., Usman, K., Alsafran, M. (2024) RuBisCO: a sustainable protein ingredient for plant-based foods. Front. sustain. food syst. ISSN=2571-581X.
4. Quintieri, L., Nitride, C., De Angelis, E., Lamonaca, A., Pilolli, R., Russo, F., Monaci, L. (2023) Alternative Protein Sources and Novel Foods: Benefits, Food Applications and Safety Issues. Nutrients. 15(6):1509.
5. Giardina, C., Pelizzola, V., Avalli, A., Iametti, S., & Cattaneo, T. M. P. (2004). Functional properties of milk protein hydrolysates obtained by controlled enzymatic hydrolysis. Milchwissenschaft, 59(9-10), 476-479.)

6. Mcclements, D. J. (2007). Critical review of techniques and methodologies for characterization of emulsion stability. Critical reviews in food science and nutrition, 47(7), 611-649).
7. Leela, G., Dayana, J., Monisha, S., Irudaya, I., Anitha, A., & Rosaline, J. V. (2016). Studies on phytochemical, nutritional analysis and screening of in vitro biological activities of Melia dubia leaf extract. Int. J. Sci. Eng. Res, 7(8), 56-68.
8. Rao, R. A. K., & Kashifuddin, M. (2016). Adsorption studies of Cd (II) on Ball Clay: comparison with other natural clays. Arabian Journal of Chemistry, 9, S1233-S1241.
9. Sharuddin, S. D. A., Abnisa, F., Daud, W. M. A. W., & Aroua, M. K. (2016). A review on pyrolysis of plastic wastes. Energy conversion and management, 115, 308-326.

,
Claims:1. A sustainable method of isolating highly pure RuBisCO protein from plant leaves the method comprising the steps of:
a) homogenizing cleaned plant leaves by using water with pH adjusted to 8.0-8.3; with leaf: water ratio as 1: 2 (w:v) to make a leaf slurry;
b) sieving the leaf slurry obtained in step (a) and collecting the filtrate and the fibre left in the sieve;
c) centrifuging the filtrate from step (b) and collecting supernatant-1 and pellet-1;
d) subjecting the supernatant-1 to thermal coagulation at 42-47°C;
e) centrifuging the thermal coagulated liquid from step (d) and collecting both supernatant-2 and pellet-2, wherein pellet-2 comprises 60-74% pure RuBisCO protein;
f) subjecting the supernatant-2 from step (e) to acid precipitation at pH 4.5; and
g) centrifuging the acid precipitated supernatant-2 from step (f) to obtain pellet-3, wherein pellet-3 comprises 75-80% pure RuBisCO protein.
2. The method of claim 1, wherein the plant leaves are from Spinach, sweet potato, broccoli, cauliflower, cabbage, banana, or papaya plants.
3. The method of claim 1, wherein the homogenization of the plant leaves in step (a) is done by mechanical means.
4. The method of claim 1, wherein the sieving in step (b) is done with a sieve with 400 mesh size.
5. The method of claim 1, wherein the centrifugation in step (c) to collect the supernatant-1 is done at 12000-16,000g for 15-20 mins at 2-8°C.
6. The method of claim 1, wherein the thermal coagulation of the supernatant-1 in step (d) is done by incubating it at 42-47°C for 10 mins and cooling down to RT.
7. The method of claim 1, wherein the centrifugation of the thermal coagulated liquid in step (e) is done at 12000-16000g for 15-20mins at 2-8°C.
8. The method of claim 1, wherein the acid precipitation of the supernatant-2 in step (f) is done with 1N HCl.
9. The method of claim 1, wherein the acid precipitation of the supernatant-2 in step (f) is done for 8-12 hours .
10. The method of claim 1, wherein the centrifugation of the acid precipitated supernatant-2 is centrifuged at 12000-16000g for 15-20mins rpm at 4°C.
11. The method of claim 1, wherein both the pellet-2 and the pellet-3 comprise high purity RuBisCO protein.
12. The method of claim 1, wherein the purified Rubisco protein in pellet-2 and pellet-3 is free of toxins, anti-nutritional substances and heavy metals.
13. The method of claim 1, wherein the pellet-1 collected after step (c) comprises 30-35% pure Rubisco protein.
14. The method of claim 1, wherein the fibre left in the sieve after homogenization and filtration of the plant leaf slurry in step (b) is also collected and comprises 15-18% pure RuBisCO protein.