Preparation process of tobacco soluble protein by mild and efficient extraction in aqueous phase

Abstract

The application relates to the technical field of biological chemical industry, and discloses a preparation process for mild and efficient extraction of tobacco soluble protein in an aqueous phase, which comprises the following steps: S1, drying and crushing waste fresh tobacco leaves, and screening the tobacco leaves through a 40-60 mesh screen to obtain tobacco leaf powder; S2, mixing the tobacco leaf powder with an alkaline buffer solution with a pH of 8.0-9.0 at a ratio of 1:15-25, homogenizing, and stirring and extracting the mixture at 35-45 DEG C for 2-3 hours; S3, centrifugally separating the extraction liquid, and collecting supernatant; S4, decolorizing the supernatant through a macroporous adsorption resin column, and collecting effluent; S5, adding acid to the effluent to adjust the pH to 3.0-4.0, centrifuging after standing, and collecting precipitate; S6, washing the precipitate with purified water, and dissolving the precipitate in water with a pH of 7.5-8.5 to obtain a protein solution; and S7, freeze-drying the protein solution to obtain tobacco soluble protein powder. Through control of the extraction temperature and the extraction time, the application realizes efficient dissolution of the tobacco soluble protein in a mild aqueous phase system, and solves the problems of protein denaturation and low extraction rate caused by high temperature in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a mild and efficient aqueous extraction process for preparing tobacco soluble proteins. Background Technology

[0002] Tobacco is an annual herbaceous economic crop belonging to the Solanaceae family and the Nicotiana genus. my country has consistently ranked first in the world in terms of planting scale and output. Throughout the entire tobacco industry chain, from planting and harvesting to curing, re-drying, and cigarette production, a large amount of waste tobacco leaves, stems, dust, and broken tobacco sheets are generated. This waste is unsuitable for cigarette production due to its substandard appearance and specifications, and the annual amount generated is enormous. For a long time, large quantities of tobacco waste have been disposed of through on-site landfill, incineration, or simple stockpiling. This not only results in a serious waste of high-quality biomass resources but also breeds mold, produces unpleasant odors, and causes soil, water, and air pollution, increasing the environmental disposal pressure on the tobacco industry and hindering its green and sustainable development.

[0003] The internal organic chemical composition of tobacco plants is complex, encompassing two major categories of active substances: macromolecules and small molecules. Among all components, protein is the most abundant and has the greatest potential for utilization among high-quality active components in tobacco. The protein content varies significantly among different tobacco varieties, with Burley tobacco containing 20.48%–21%. Compared to conventional plant proteins such as soybean protein and cereal protein, tobacco protein has a balanced amino acid profile, containing all the essential amino acids necessary for human and animal growth. Its nutritional value is close to that of high-quality milk protein, offering advantages such as easy digestibility, high absorption, and low allergenicity. Furthermore, tobacco protein possesses unique antioxidant, emulsifying, film-forming, and carrier coating properties, showing broad application prospects in feed processing, food ingredients, biological carriers, functional new materials, and biopharmaceutical excipients.

[0004] Currently, extensive research has been conducted both domestically and internationally on the resource utilization technology of tobacco waste, but the overall utilization level is low, and there are significant shortcomings in industrial application. The mainstream utilization methods currently focus on low-end, extensive processing, mainly including nicotine extraction, high-temperature activated carbon preparation, organic fertilizer composting, and biomass fuel combustion. These technologies generally suffer from low added value, cumbersome processes, high energy consumption, significant pollution, and low resource utilization rates. Existing tobacco protein extraction technologies have the following main problems: Existing extraction processes often employ high-temperature (above 60°C) or strong alkaline (pH>10) conditions, which result in severe thermal denaturation of proteins, destruction of their natural structure, and extraction rates typically below 50%, while also deteriorating the functional properties of the obtained proteins.

[0005] Some processes use organic solvents such as acetone and methanol for degreasing or precipitation, which poses a risk of solvent residue, does not meet the safety requirements for food-grade proteins, and increases production costs and environmental pressure.

[0006] The lack of an effective decolorization and impurity removal step during the extraction process results in proteins with dark color, high impurity content, poor purity and sensory quality, which limits their application in the food and high-end materials fields.

[0007] Existing processes produce protein particles with large sizes (micrometers) and small specific surface areas, which are not conducive to their use as nanodelivery carriers or microcapsule wall materials, thus limiting their functional application expansion.

[0008] In view of this, we propose a mild and efficient aqueous extraction process for preparing tobacco soluble proteins. Summary of the Invention

[0009] The purpose of this invention is to provide a mild and efficient aqueous extraction process for tobacco soluble proteins to solve the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A mild and efficient aqueous extraction process for preparing tobacco-soluble proteins, the method comprising: S1. Dry and pulverize the waste fresh tobacco leaves, and pass them through a 40-60 mesh sieve to obtain tobacco powder; S2. Mix the tobacco powder obtained in step S1 with an alkaline buffer solution with a pH of 8.0 to 9.0 at a material-to-liquid ratio of 1:15 to 1:25. After homogenization, stir and extract at 35 to 45°C for 2.0 to 3.0 hours to obtain the extract. S3. Centrifuge the extract obtained in step S2 and collect the supernatant; S4. Decolorize the supernatant obtained in step S3 by passing it through a macroporous adsorption resin column and collect the effluent. S5. Add acid to the effluent obtained in step S4 to adjust the pH to 3.0-4.0, let it stand, centrifuge, and collect the precipitate; S6. Wash the precipitate obtained in step S5 with purified water, and then redissolve it in water with a pH of 7.5 to 8.5 to obtain a protein solution; S7. Freeze-dry the protein solution obtained in step S6 to obtain tobacco soluble protein powder.

[0011] Preferably, the alkaline buffer in step S2 is a phosphate buffer or a Tris-HCl buffer with a pH of 8.5; the material-to-liquid ratio is 1:20; the stirring extraction time is 2.5 hours; and the extraction temperature is 40°C.

[0012] Preferably, the homogenization process in step S2 uses a high-speed homogenizer with a rotation speed of 8,000 to 12,000 rpm and a homogenization time of 3 to 8 minutes.

[0013] Preferably, the centrifugal force for centrifugation in step S3 is 8,000 to 12,000 × g, and the centrifugation time is 15 to 25 minutes.

[0014] Preferably, the macroporous adsorption resin in step S4 is D3520 type macroporous adsorption resin; before decolorization, the resin is first soaked and rinsed with anhydrous ethanol and distilled water in sequence until there is no alcohol odor, and then the resin column is equilibrated with buffer solution with pH 8.0-9.0; the sample loading flow rate is 1-3 BV / h, and the sample loading temperature is room temperature; after the sample loading is completed, the resin is eluted with 2-3 column volumes of buffer solution with pH 8.0-9.0, and the eluent is combined with the main decolorized effluent.

[0015] Preferably, the acid in step S5 is hydrochloric acid or citric acid; the standing time is 10 to 30 minutes; the centrifugal force is 8000 to 12000 × g, and the centrifugation time is 15 to 25 minutes.

[0016] Preferably, the washing in step S6 uses an equal volume of purified water, and the washing is performed 1 to 3 times; the concentration of the reconstituted protein solution is 5 to 20 mg / mL.

[0017] Preferably, the freeze-drying in step S7 includes: pre-freezing at -20°C for 12 hours, and then freeze-drying in stages for 48 hours under conditions of vacuum degree ≤10 Pa and cold trap temperature -50°C, wherein the first 24 hours are sublimation drying and the last 24 hours are desorption drying.

[0018] Preferably, the extraction rate of the obtained tobacco soluble protein is ≥68%, and the purity is ≥85%; the protein particles are nearly spherical with a particle size of 40-50 nm, and the surface has a concave structure and abundant pores.

[0019] Preferably, the amino acid composition of the obtained tobacco soluble protein contains essential amino acids accounting for ≥50% of the total amino acids, and contains at least four of the following: aspartic acid, glutamic acid, leucine, arginine, and proline.

[0020] By employing the above technical solution, the present invention provides a mild and efficient aqueous extraction process for preparing tobacco soluble proteins. It possesses at least the following beneficial effects: This invention achieves highly efficient dissolution of tobacco-soluble proteins in a mild aqueous system by controlling the extraction temperature at 35–45°C (preferably 40°C), using an alkaline buffer solution with a pH of 8.0–9.0, and an extraction time of 2.0–3.0 hours. The extraction rate can reach up to 70.58%. Compared with existing high-temperature (>60°C) extraction processes, this invention avoids protein thermal denaturation and aggregation precipitation, solving the problems of protein denaturation and low extraction rates caused by high temperatures in existing technologies. Furthermore, it eliminates the use of organic solvents such as acetone and methanol throughout the process, thus addressing the risks of organic solvent residues and environmental concerns.

[0021] By employing D3520 macroporous adsorption resin to decolorize the extract, combined with low-speed loading and buffer elution, chlorophyll and polyphenolic colored impurities were effectively removed, resulting in a light-colored protein with high purity (≥85%). Compared to existing processes that lack an effective decolorization step, leading to dark protein color and high impurity content, this invention solves the problem of poor sensory quality of proteins that cannot meet food-grade requirements, thus broadening the application of proteins in food ingredients and high-end materials.

[0022] Nanoscale tobacco soluble protein particles (40-50 nm in diameter) with abundant surface depressions and pores were obtained by combining acid precipitation (pH 3.0-4.0) with freeze-drying. Compared with micron-sized protein particles obtained by existing processes, the protein obtained by this invention has a larger specific surface area and stronger adsorption capacity, solving the problems of excessive size and insufficient performance of proteins in functional applications such as nanodelivery carriers and microcapsule wall materials, and providing a new technical path for the high-value utilization of tobacco protein.

[0023] By optimizing the material-to-liquid ratio (1:20) and extraction time (2.5 hours), a balance between protein dissolution efficiency and process cost was achieved. An excessively high material-to-liquid ratio increases the workload of subsequent decolorization and concentration, while an excessively low ratio results in insufficient protein dissolution. Conversely, an excessively long extraction time leads to protein degradation (extraction rate drops to 55.42% after 3.0 hours), while an excessively short extraction time results in insufficient dissolution. This invention precisely controls these two parameters, solving the problem of resource waste or protein degradation caused by improper parameters in existing processes.

[0024] Amino acid composition analysis showed that the obtained tobacco soluble protein contained all eight essential amino acids required by the human body, accounting for 54.07% of the total amino acids. Among them, glutamic acid content was as high as 21.81 mg / g, and hydrophobic amino acids such as leucine and valine were abundant. Compared with soybean protein and cereal protein, the tobacco protein obtained by this invention has a more balanced amino acid ratio and higher nutritional value, solving the problem of certain essential amino acid deficiencies in existing plant protein sources and providing a high-quality alternative protein raw material for the feed and food industries. Attached Figure Description

[0025] The accompanying drawings, which are provided to further illustrate the invention, constitute a part of this application: Figure 1 This is a schematic diagram of the overall process for the preparation of a mild and efficient aqueous extraction process for soluble tobacco protein according to the present invention. Figure 2 The figure shows the effect of different stirring times on the extraction rate of tobacco soluble protein in a mild and efficient aqueous extraction process of tobacco soluble protein according to the present invention. Figure 3This figure shows the effect of different material-liquid ratios on the extraction rate of tobacco soluble protein in a mild and efficient aqueous extraction process for tobacco soluble protein according to the present invention. Figure 4 This figure shows the effect of different pH values ​​on the extraction rate of tobacco soluble protein in a mild and efficient aqueous extraction process for tobacco soluble protein according to the present invention. Figure 5 This figure shows the effect of different temperatures on the extraction rate of tobacco soluble protein in a mild and efficient aqueous extraction process for tobacco soluble protein according to the present invention. Figure 6 This is a SEM image of tobacco protein in a mild and efficient aqueous extraction process for preparing soluble tobacco protein according to the present invention. Figure 7 This is a spectral characterization diagram of tobacco soluble protein in a mild and efficient aqueous extraction process for preparing tobacco soluble protein according to the present invention. Figure 8 This is a gel electrophoresis characterization image of tobacco soluble protein in a mild and efficient aqueous extraction process for preparing tobacco soluble protein according to the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Please see Figure 1 , Figure 1 This is a schematic diagram of the overall process for a mild and efficient aqueous extraction preparation process of tobacco soluble protein according to the present invention. The present invention provides a mild and efficient aqueous extraction preparation process of tobacco soluble protein, comprising: S1. Dry and pulverize the waste fresh tobacco leaves, and pass them through a 40-60 mesh sieve to obtain tobacco powder; It should be noted that the selection of discarded fresh tobacco leaves mainly consists of the lower leaves, diseased leaves, and upper leaves after topping, which are removed after harvesting. These leaves have a high protein content and have not yet undergone severe browning. The drying temperature should ideally be controlled at 60℃. Excessive temperature will intensify the Maillard reaction, causing cross-linking of proteins and reducing sugars, thus reducing the subsequent extraction rate; excessively low temperature will prolong the drying time and easily promote microbial growth. The choice of sieve mesh size is crucial: a 40-mesh sieve has too large an aperture, allowing impurities such as fiber bundles to easily enter; a 60-mesh sieve has too small an aperture, making the powder too fine and causing difficulties in solid-liquid separation during subsequent centrifugation. A 50-mesh sieve is preferred, balancing extraction efficiency and ease of subsequent operation. The pulverized tobacco powder should be sealed and stored away from light to prevent moisture absorption and oxidative deterioration.

[0028] S2. Mix the tobacco powder obtained in step S1 with an alkaline buffer solution with a pH of 8.0 to 9.0 at a material-to-liquid ratio of 1:15 to 1:25. After homogenization, stir and extract at 35 to 45°C for 2.0 to 3.0 hours to obtain the extract. It should be noted that the four key parameters (pH, solid-liquid ratio, temperature, and time) need to be controlled in a coordinated manner. The pH selection of 8.0–9.0 is based on the isoelectric point characteristics of tobacco-soluble proteins (mainly Rubisco, i.e., ribulose-1,5-bisphosphate carboxylase / oxygenase). Within this pH range, the protein has a negative net charge, enhancing intermolecular electrostatic repulsion and facilitating dissolution from the cell matrix. A solid-liquid ratio of 1:15–1:25 balances mass transfer efficiency with the subsequent processing load: a ratio that is too low (e.g., 1:10) results in a viscous slurry, limiting protein dissolution; a ratio that is too high (e.g., 1:30) leads to excessive dilution, significantly increasing the amount of chemicals and time required for subsequent decolorization and acid precipitation. Homogenization ensures thorough cell wall disruption, releasing intracellular proteins. A rotation speed of 8000–12000 rpm and a time of 3–8 minutes ensure effective disruption without excessive heat leading to protein denaturation. The extraction temperature of 35-45℃ is the result of a precise trade-off: below 35℃, insufficient molecular thermal motion leads to a slow protein dissolution rate; above 45℃, protease activity increases, and Rubisco begins to undergo thermal denaturation; above 60℃, the extraction rate drops sharply. An extraction time of 2.0-3.0 hours represents the balance between dissolution kinetics and protein stability, with 2.5 hours being optimal. Beyond 3 hours, the protein degradation rate exceeds the dissolution rate, resulting in a decrease in extraction rate.

[0029] S3. Centrifuge the extract obtained in step S2 and collect the supernatant; It should be noted that the purpose of centrifugation is to remove unbroken cell debris, fibrous residue, and insoluble impurities. A centrifugal force of 8000–12000 × g and a time of 15–25 minutes are recommended. Under these conditions, particles larger than 1 μm in diameter can be effectively settled, while soluble proteins remain in the supernatant. If the centrifugal force is too low or the time is too short, fine suspended matter will be mixed into the supernatant, affecting the subsequent decolorization efficiency and protein purity. If the centrifugal force is too high or the time is too long, some protein aggregates may settle, resulting in yield loss. The supernatant after centrifugation is yellowish-brown to brownish-yellow, which is the result of the combined color development of chlorophyll, polyphenols, and a small amount of nicotine, requiring further decolorization.

[0030] S4. Decolorize the supernatant obtained in step S3 by passing it through a macroporous adsorption resin column and collect the effluent. It should be noted that the macroporous adsorption resin D3520 has a non-polar polystyrene framework, exhibiting strong physical adsorption capacity for moderately polar pigment molecules such as chlorophyll and polyphenols, while showing almost no adsorption for large molecules such as proteins, thus achieving selective decolorization. Resin pretreatment must be thorough: soaking in anhydrous ethanol for 24 hours dissolves residual oligomers and porogens from the resin synthesis process; washing with distilled water until no alcohol odor remains eliminates interference from ethanol on the protein solution. The pH of the equilibration buffer should be consistent with the pH of the supernatant (8.0~9.0) to avoid sudden pH changes that could lead to protein precipitation. A loading flow rate of 1~3 BV / h is considered low-speed operation, aiming to prolong the contact time between the sample and the resin, increasing the pigment adsorption capacity; a flow rate that is too fast will result in premature breakthrough and incomplete decolorization. After loading, elution with 2~3 column volumes of the same pH buffer washes away proteins trapped in the resin interstices, reducing yield loss. Combining the main decolorization effluent and the eluent yields a protein solution with a significantly lighter color.

[0031] S5. Add acid to the effluent obtained in step S4 to adjust the pH to 3.0-4.0, let it stand, centrifuge, and collect the precipitate; It should be noted that the isoelectric point (pI) of tobacco soluble proteins (especially Rubisco) is approximately 4.5–5.5. However, in actual systems, due to the complex interaction between proteins and other molecules (such as polyphenols and polysaccharides), the optimal precipitation pH is slightly lower than the theoretical isoelectric point, falling within the range of 3.0–4.0. When adjusting the pH, the acid solution should be added slowly with continuous stirring to prevent localized over-acidity that could lead to irreversible protein denaturation. Hydrochloric acid is a strong inorganic acid, resulting in rapid precipitation with a small dosage; citric acid is a weak organic acid with strong buffering capacity and a gentler effect on protein conformation, and can be chosen based on the intended use. Allowing the mixture to stand for 10–30 minutes allows the protein flocs to grow sufficiently, facilitating centrifugation. The precipitate obtained after centrifugation is grayish-white to pale yellow, which is the crude target protein. The supernatant mainly contains residual nicotine, small-molecule sugars, and inorganic salts.

[0032] S6. Wash the precipitate obtained in step S5 with purified water, and then redissolve it in water with a pH of 7.5 to 8.5 to obtain a protein solution; It should be noted that the purpose of washing is to remove residual acid, salts, and small amounts of pigment carried in the precipitate. Wash 1-3 times with an equal volume of purified water, centrifuging to collect the precipitate after each wash. Too many washes will lead to increased protein loss, while too few washes will leave residual impurities affecting the final purity. The pH for reconstitution should be 7.5-8.5, consistent with the principle that this protein has optimal solubility under slightly alkaline conditions. The protein concentration after reconstitution should be controlled at 5-20 mg / mL: too low a concentration will result in low efficiency and high energy consumption for subsequent freeze-drying; too high a concentration will result in high solution viscosity, which is not conducive to water sublimation during freeze-drying and tends to form a glassy state rather than a loose and porous cake structure.

[0033] S7. Freeze-dry the protein solution obtained in step S6 to obtain tobacco soluble protein powder; It should be noted that freeze-drying is the optimal drying method for preserving the natural structure of proteins. A pre-freezing temperature of -20°C for 12 hours allows the protein solution to completely crystallize and solidify, forming a uniform ice crystal framework. A vacuum degree ≤10 Pa ensures sufficient sublimation driving force, and a cold trap temperature of -50°C effectively captures the sublimated water vapor. The scientific basis for the segmented freeze-drying strategy is as follows: the first 24 hours are the sublimation drying stage, mainly removing free water; this stage should maintain a low temperature to prevent collapse. The following 24 hours are the desorption drying stage, mainly removing bound water; the temperature can be appropriately increased (through partition heating) to improve efficiency. The resulting protein powder is a pale yellow, loose, lumpy mass that crumbles easily upon light pressure, exhibits good resolubility, and can be directly used for subsequent application research or long-term storage.

[0034] The alkaline buffer solution in step S2 is phosphate buffer or Tris-HCl buffer with a pH of 8.5; the material-to-liquid ratio is 1:20; the stirring extraction time is 2.5 hours; and the extraction temperature is 40°C. It should be noted that both phosphate buffer and Tris-HCl buffer are commonly used pH-stabilized systems in biochemical experiments. Phosphate buffer has a large buffering capacity around pH 8.5, and phosphate ions have little effect on protein conformation; Tris-HCl buffer does not contain metal ions and is suitable for applications that may require subsequent metal ion chelation. pH 8.5 is the optimal compromise between extraction rate and protein denaturation: single-factor experiments showed that the extraction rate reached 69.12% at pH 8.5, which was not significantly different from 69.36% at pH 9.0. However, pH 9.0 is more alkaline and may cause side reactions such as deamidation of protein side chains and disulfide bond breakage. A material-to-liquid ratio of 1:20, an extraction time of 2.5 hours, and a temperature of 40℃ were the optimal values ​​confirmed by single-factor experiments, under which the extraction rate reached 70.58%.

[0035] The homogenization process described in step S2 uses a high-speed homogenizer with a rotation speed of 8000-12000 rpm and a homogenization time of 3-8 minutes; It should be noted that the high-speed homogenizer works by generating high-speed shearing force, impact force, and cavitation effect through the rotor-stator system, which fully disperses the tobacco powder in the liquid phase and breaks down cell walls. A rotation speed of 8000-12000 rpm is considered moderate intensity, effectively breaking down cells and releasing proteins without generating excessive heat due to excessive shearing, which could lead to protein denaturation. The homogenization time of 3-8 minutes should be adjusted flexibly according to the feed rate and slurry viscosity: for large feed rates or viscous slurries, the time can be extended to 8 minutes, and vice versa. The slurry temperature will rise during homogenization; if it exceeds 45℃, an ice bath should be used for cooling. After homogenization, the slurry should be uniformly turbid with no visible powder particles.

[0036] The centrifugal force for centrifugation in step S3 is 8000-12000×g, and the centrifugation time is 15-25 minutes; It should be noted that the selection of centrifugal force must take into account the conversion relationship between the centrifuge rotor radius and rotational speed. The rotational speed corresponding to 8000~12000g is typically between 6000~10000rpm (depending on the rotor radius), under which particles with a diameter greater than 1μm can be effectively settled. A centrifugation time of 15~25 minutes is sufficient for the solid phase to form a compact precipitate, and the supernatant to be clear and transparent. If the supernatant is still cloudy after centrifugation, it indicates that the centrifugal force or time is insufficient, and the parameters can be appropriately increased; if the precipitate is too compact and the supernatant is difficult to pour out, it indicates that the centrifugal force or time is too large, and it can be appropriately reduced. The centrifugation temperature is recommended to be controlled between 4~10℃, as low temperatures can reduce protease activity and decrease protein degradation.

[0037] The macroporous adsorption resin mentioned in step S4 is D3520 type macroporous adsorption resin; before decolorization, the resin is first soaked and rinsed with anhydrous ethanol and distilled water in sequence until there is no alcohol odor, and then the resin column is equilibrated with buffer solution with pH 8.0-9.0; the sample loading flow rate is 1-3 BV / h, and the sample loading temperature is room temperature; after the sample loading is completed, the resin is eluted with 2-3 column volumes of buffer solution with pH 8.0-9.0, and the eluent is combined with the main decolorized effluent; It is worth noting that the specific surface area of ​​the D3520 macroporous adsorption resin is approximately 400~600 m². 2 The resin has an average pore size of approximately 8-10 nm and exhibits optimal adsorption for moderately polar pigment molecules with molecular weights ranging from 300 to 2000 Da. Sufficient pretreatment is essential before use; otherwise, residual porogens (such as toluene or xylene) will contaminate the protein solution. The standard for determining resin cleanliness is that the eluent has an ultraviolet absorbance of less than 0.05 at 210 nm and exhibits no odor. During sample loading, the color change of the effluent should be checked periodically. When the effluent color darkens significantly, it indicates that the resin adsorption is approaching saturation, and sample loading should be stopped immediately for regeneration. Resin regeneration can be achieved by soaking in 0.5 mol / L NaOH solution for 2 hours, then washing with distilled water until neutral, followed by soaking in anhydrous ethanol. It can be reused 5-10 times. Combining the eluent with the main decolorizing effluent will slightly reduce the protein concentration, but this will not affect the subsequent acid precipitation effect.

[0038] The acid mentioned in step S5 is hydrochloric acid or citric acid; the standing time is 10 to 30 minutes; the centrifugal force is 8000 to 12000 × g, and the centrifugation time is 15 to 25 minutes; It is worth noting that hydrochloric acid is a strong acid, and its pH response is rapid upon addition, but it carries a high risk of localized over-acidity. Therefore, it is recommended to add it dropwise with vigorous stirring. Citric acid is a tribasic weak acid with a buffering effect, which can make the precipitation process gentler and preserve the protein conformation more intact, but the amount used is relatively large. The principle for determining the settling time is: when obvious flocculent matter appears in the solution and begins to settle, continue settling for another 5-10 minutes. If the settling time is too long, the precipitated protein may redissolve or become contaminated with microorganisms. After centrifugation, the precipitate can be collected by decantation, discarding the supernatant while being careful not to disturb the precipitate layer. If the amount of precipitate is small, the supernatant can be carefully removed with a pipette.

[0039] The washing in step S6 uses an equal volume of purified water, and the washing is performed 1 to 3 times; the concentration of the reconstituted protein solution is 5 to 20 mg / mL. It is important to note that the conductivity of the purified water used for washing should be ≤1 μS / cm to avoid introducing additional ions. During the first wash, the precipitate may partially dissolve, resulting in a slightly turbid supernatant, which is normal. The supernatant should be mostly clear after the second wash; after the third wash, the supernatant should be colorless and transparent. If the supernatant still has a noticeable color after the third wash, it indicates that decolorization before acid precipitation was incomplete, and the decolorization steps should be checked retrospectively. For reconstitution, the precipitate can be first mixed with a small amount of water at pH 8.0 and ground into a paste, then gradually diluted with water to the target concentration. This method can accelerate the dissolution rate. For protein concentration determination, the Bradford method or BCA method is recommended for its speed and accuracy.

[0040] The freeze-drying described in step S7 includes: first pre-freezing at -20°C for 12 hours, and then freeze-drying in stages for 48 hours under conditions of vacuum degree ≤10 Pa and cold trap temperature -50°C, wherein the first 24 hours are sublimation drying and the last 24 hours are desorption drying; It is worth noting that a pre-freezing temperature of -20℃ and a time of 12 hours are suitable for commonly sized freeze-drying bottles (with a liquid volume not exceeding 1 / 3 of the bottle's volume). If the liquid volume is large or the container shape is irregular, the pre-freezing time should be appropriately extended to ensure complete freezing. During the sublimation drying stage, the material temperature should always be kept below the eutectic point (the eutectic point of protein solutions is usually between -15℃ and -25℃), which can be achieved by adjusting the separator temperature. During the desorption drying stage, the separator temperature can be gradually increased to 20~30℃ to provide the energy required for desorption of bound water, but it should not exceed 40℃, otherwise the protein may denature. The criteria for judging the end of freeze-drying are: the material temperature and the separator temperature tend to be consistent, and the vacuum degree stabilizes below 1 Pa. After freeze-drying, dry nitrogen or argon should be immediately introduced and the bottle sealed to prevent moisture absorption.

[0041] The extraction rate of soluble tobacco protein was ≥68%, and the purity was ≥85%. The protein particles were nearly spherical with a particle size of 40-50 nm and had a concave structure and abundant pores on the surface. It is worth noting that an extraction rate of ≥68% represents a stable performance under optimal process conditions. In actual production, this may fluctuate slightly due to batch variations in raw materials, but should not be lower than 65%. A purity of ≥85% is based on the Kjeldahl nitrogen determination method. If the Lowry method or BCA method is used, the value may differ due to variations in the response coefficients of different methods to proteins. The nanoscale particle size (40-50 nm) of the protein particles is a major feature of this process, thanks to the mild extraction conditions and rapid solidification during freeze-drying, which prevents excessive aggregation of protein molecules. The formation of surface depressions and pores is related to the voids left by ice crystal sublimation during freeze-drying. These structures endow the protein with a large specific surface area (measured at approximately 50-80 m²). 2 / g), which is beneficial for subsequent applications as an adsorbent or delivery carrier.

[0042] The amino acid composition of the obtained tobacco soluble protein contains ≥50% essential amino acids, and contains at least four of the following: aspartic acid, glutamic acid, leucine, arginine, and proline. It is worth noting that an essential amino acid content of ≥50% is a core indicator for measuring the nutritional value of protein. The ideal protein profile recommended by FAO / WHO has an essential amino acid content of approximately 40%, and the tobacco protein obtained in this invention significantly surpasses this standard. Aspartic acid and glutamic acid are acidic amino acids, giving the protein good water solubility and pH buffering capacity; leucine is a key signaling molecule in muscle protein synthesis and has outstanding value in sports nutrition and feed; arginine is a precursor to nitric oxide synthesis and has a regulatory effect on cardiovascular health and immune function; the pyrrolidine ring structure of proline gives the protein chain rigidity, which helps to form a stable three-dimensional conformation.

[0043] Example 1 Using waste fresh tobacco leaves as raw material, the optimal extraction process parameters were determined through single-factor experiments.

[0044] (1) Raw material preparation: Dry the waste fresh tobacco leaves at 60°C to constant weight, crush them with a pulverizer and pass them through a 50-mesh sieve to obtain tobacco powder, which is then sealed and stored for later use.

[0045] (2) Optimization of extraction time: Weigh 10g of tobacco powder and add 200mL of pH 8.5 phosphate buffer (solid-to-liquid ratio 1:20). Homogenize at 10000rpm for 5 minutes using a high-speed homogenizer. Place the homogenate in a 40℃ water bath and extract with magnetic stirring for 1.0h, 1.5h, 2.0h, 2.5h, and 3.0h respectively. After extraction, centrifuge at 10000×g for 20 minutes and collect the supernatant. Determine the protein content according to the Kjeldahl method and calculate the extraction rate. The results are as follows: Figure 2As shown, the extraction rate first increases and then decreases with time, reaching a peak of 70.58% at 2.5 h and decreasing to 55.42% at 3.0 h. Therefore, the optimal extraction time is determined to be 2.5 hours.

[0046] (3) Optimization of the material-liquid ratio: With a fixed extraction time of 2.5 h, a temperature of 40℃, and a pH of 8.5, the material-liquid ratios were set to 1:10, 1:15, 1:20, 1:25, and 1:30, respectively. The results are as follows: Figure 3 As shown, the extraction rate first increases and then decreases with the increase of the material-to-liquid ratio, reaching a maximum of 70.58% at a ratio of 1:20 and decreasing to 62.51% at a ratio of 1:25. Therefore, the optimal material-to-liquid ratio is determined to be 1:20.

[0047] (4) Optimization of pH for alkaline solubility: With a fixed extraction time of 2.5 h, temperature of 40℃, and material-to-liquid ratio of 1:20, the pH values ​​were set to 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. The results are as follows: Figure 4 As shown, the extraction rate gradually increases with increasing pH, reaching a maximum of 69.36% at pH 9.0, but already reaching 69.12% at pH 8.5. Considering that excessively high pH may cause protein denaturation, pH 8.5 is the optimal choice.

[0048] (5) Optimization of extraction temperature: With a fixed extraction time of 2.5 h, a material-to-liquid ratio of 1:20, and pH of 8.5, the temperatures were set at 0℃, 20℃, 40℃, 60℃, and 80℃, respectively. The results are as follows: Figure 5 As shown, the extraction rate first increases and then decreases with increasing temperature, reaching a maximum of 70.27% at 40℃, decreasing to 64.88% at 60℃, and further decreasing to 52.31% at 80℃. Therefore, the optimal extraction temperature is determined to be 40℃.

[0049] Based on the results of the single-factor experiments above, the optimal extraction process parameters were determined to be: material-to-liquid ratio 1:20, extraction time 2.5 hours, extraction temperature 40℃, and alkali-soluble pH 8.5.

[0050] Example 2 In this embodiment, the extraction was scaled up according to the optimal process parameters determined in Example 1, and the obtained protein was systematically characterized.

[0051] (1) Extraction and preparation: Weigh 50g of tobacco powder, add 1000mL of pH 8.5 phosphate buffer (solid-to-liquid ratio 1:20), and homogenize at 10000rpm for 5 minutes. Transfer the homogenate to a 40℃ water bath and extract with magnetic stirring for 2.5 hours. Centrifuge the extract at 10000×g for 20 minutes and collect the supernatant.

[0052] (2) Decolorization treatment: D3520 macroporous adsorption resin was packed into a glass chromatography column (column volume 100 mL), soaked in anhydrous ethanol for 24 hours, washed with distilled water until no alcohol odor remained, and then equilibrated with phosphate buffer at pH 8.5. The supernatant was loaded at a flow rate of 2 BV / h at room temperature, and the eluent was collected. After loading, the resin was eluted with 2 column volumes of pH 8.5 buffer, and the eluent and main decolorized eluent were combined and the total volume was recorded.

[0053] (3) Acid precipitation purification: Slowly add 1 mol / L HCl to the decolorized effluent while stirring, adjust the pH to 3.5, and let stand for 20 minutes. Then centrifuge at 10000×g for 20 minutes, discard the supernatant, and collect the precipitate.

[0054] (4) Washing and redissolving: Wash the precipitate once with an equal volume of distilled water, and then centrifuge to collect the precipitate. Redissolve the precipitate in an appropriate amount of water at pH 8.0 to make the protein concentration approximately 10 mg / mL.

[0055] (5) Freeze-drying: Place the protein solution in a -20℃ freezer for 12 hours. After it is completely frozen, transfer it to a vacuum freeze dryer with a vacuum degree ≤10 Pa and a cold trap temperature of -50℃. First, sublimate and dry for 24 hours, then decompose and dry for 24 hours to obtain a light yellow loose powder. Store it in a sealed container away from light.

[0056] (6) Physicochemical property characterization: Extraction rate determination: The extraction rate was 70.38% according to the Kjeldahl nitrogen determination method.

[0057] Purity determination: The Kjeldahl method was used, and the protein purity was 87.2%.

[0058] Scanning electron microscope (SEM): such as Figure 6 As shown, the protein particles are nearly spherical with an uneven surface and a large number of irregular depressions. The particle size is concentrated in the range of 40-50 nm, and the particles are partially adhered to each other, forming abundant pores.

[0059] FTIR (Full Infrared Spectroscopy): such as Figure 7 As shown, 3413cm -1 The broad peaks at 2932 cm⁻¹ indicate the presence of a large hydrogen bond network, representing the stretching vibrations of NH and OH. -1 It is an antisymmetric stretching vibration of -CH2-; 1600~1700cm -1 The band is amide I (-C=O- stretching vibration), indicating that the protein's secondary structure is well preserved.

[0060] Gel electrophoresis (SDS-PAGE): such as Figure 8As shown, clear bands are visible in the high molecular weight region (105–140 kDa), corresponding to Rubisco oligomers; a very coarse main band (approximately 55 kDa) appears in the 52–66 kDa range, representing the large Rubisco subunit with extremely high abundance; the three sets of parallel bands are consistent, indicating good extraction reproducibility.

[0061] (7) Amino acid composition analysis: The amino acid composition was determined using an automatic amino acid analyzer, and the results are shown in Table 1. Essential amino acids accounted for 54.07% of the total amino acids, with glutamic acid having the highest content (21.81 mg / g), followed by leucine (11.74 mg / g), proline (12.70 mg / g), and arginine (11.01 mg / g). The amino acid composition was balanced.

[0062] Table 1

[0063] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mild and efficient aqueous extraction process for preparing tobacco-soluble proteins, characterized in that, The process includes: S1. Dry and pulverize the waste fresh tobacco leaves, and pass them through a 40-60 mesh sieve to obtain tobacco powder; S2. Mix the tobacco powder obtained in step S1 with an alkaline buffer solution with a pH of 8.0 to 9.0 at a material-to-liquid ratio of 1:15 to 1:

25. After homogenization, stir and extract at 35℃ to 45℃ for 2.0 hours to 3.0 hours to obtain the extract. S3. Centrifuge the extract obtained in step S2 and collect the supernatant; S4. Decolorize the supernatant obtained in step S3 by passing it through a macroporous adsorption resin column and collect the effluent. S5. Add acid to the effluent obtained in step S4 to adjust the pH to 3.0-4.0, let it stand, centrifuge, and collect the precipitate; S6. Wash the precipitate obtained in step S5 with purified water, and then redissolve it in water with a pH of 7.5 to 8.5 to obtain a protein solution; S7. Freeze-dry the protein solution obtained in step S6 to obtain tobacco soluble protein powder.

2. The process for mild and efficient aqueous extraction of tobacco soluble protein according to claim 1, characterized in that, The alkaline buffer solution in step S2 is a phosphate buffer solution with a pH of 8.5; the material-to-liquid ratio is 1:20; the stirring extraction time is 2.5 hours; and the extraction temperature is 40°C.

3. The process for mild and efficient aqueous extraction of tobacco soluble protein according to claim 1, characterized in that, The homogenization process in step S2 uses a high-speed homogenizer with a rotation speed of 8000 rpm to 12000 rpm and a homogenization time of 3 to 8 minutes.

4. The mild and efficient aqueous extraction and preparation process for tobacco soluble protein according to claim 1, characterized in that, The centrifugal force for centrifugation in step S3 is 8000×g to 12000×g, and the centrifugation time is 15 minutes to 25 minutes.

5. The mild and efficient aqueous extraction process for preparing tobacco soluble protein according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step S4 is D3520 type macroporous adsorption resin; before decolorization, the resin is first soaked and rinsed with anhydrous ethanol and distilled water in sequence until there is no alcohol odor, and then the resin column is equilibrated with buffer solution with pH 8.0 to 9.0; the sample loading flow rate is 1 BV / h to 3 BV / h, and the sample loading temperature is room temperature; after the sample loading is completed, the resin is eluted with 2 to 3 column volumes of buffer solution with pH 8.0 to 9.0, and the eluent is combined with the main decolorized effluent.

6. The mild and efficient aqueous extraction process for preparing tobacco soluble protein according to claim 1, characterized in that, The acid mentioned in step S5 is hydrochloric acid; the standing time is 10 to 30 minutes; the centrifugal force is 8000 to 12000 × g, and the centrifugation time is 15 to 25 minutes.

7. The mild and efficient aqueous extraction process for preparing tobacco soluble protein according to claim 1, characterized in that, The washing in step S6 uses an equal volume of purified water and is performed 1 to 3 times; the concentration of the reconstituted protein solution is 5 to 20 mg / mL.

8. The mild and efficient aqueous extraction process for preparing tobacco soluble protein according to claim 1, characterized in that, The freeze-drying described in step S7 includes: first pre-freezing at -20°C for 12 hours, and then freeze-drying in stages for 48 hours under conditions of vacuum degree ≤10 Pa and cold trap temperature -50°C, wherein the first 24 hours are sublimation drying and the last 24 hours are desorption drying.

9. The mild and efficient aqueous extraction process for preparing tobacco soluble protein according to claim 1, characterized in that, The extraction rate of soluble tobacco protein was ≥68%, and the purity was ≥85%. The protein particles were nearly spherical with a particle size of 40-50 nm and had a concave structure and abundant pores on the surface.

10. The mild and efficient aqueous extraction and preparation process for tobacco soluble protein according to claim 1, characterized in that, The amino acid composition of the obtained tobacco soluble protein contains ≥50% essential amino acids, and contains at least four of the following: aspartic acid, glutamic acid, leucine, arginine, and proline.

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