Method for extracting insoluble tobacco protein, insoluble tobacco protein and application of insoluble tobacco protein

Abstract

The invention belongs to the field of food, and particularly relates to a method for extracting insoluble tobacco protein, which comprises the following steps: (1) mixing a crude product containing the insoluble tobacco protein with chlorite in a solvent, and reacting at 15-90 DEG C to obtain a reaction product; (2) carrying out centrifugal treatment on the reaction product, and collecting an upper-layer material; and (3) mixing the upper-layer material with a precipitator, carrying out solid-liquid separation, and collecting a solid-phase substance, namely the insoluble tobacco protein. The invention further relates to the obtained insoluble tobacco protein and application thereof. The insoluble tobacco protein extracted through the method is high in purity and large in brightness value and yellowness value, the total color difference change is obvious compared with a crude product, and the product quality is improved.

Description

Technical Field

[0001] The invention belongs to the field of food, and specifically relates to a method for extracting insoluble tobacco protein, and also relates to the obtained insoluble tobacco protein and the application of the insoluble tobacco protein. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is an annual herbaceous plant of the genus Nicotiana in the Solanaceae family. The organic chemical composition of tobacco is very complex, including macromolecules such as protein, fiber, pectin, starch, lignin, hemicellulose, etc., as well as small molecules such as nicotine, solanesol, caffeic acid, rutin, quercetin, vitamin B3, etc. Among them, protein content is the most abundant, such as the protein content in burley tobacco is as high as 20.48%. The nutritional value of protein in tobacco is similar to that of milk protein, and it can be used as a high-quality food source. Compared with other proteins, tobacco protein has higher nutritional value and medicinal value, and is gradually applied to the fields of feed and food. Extracting natural protein from tobacco is an important way to comprehensively utilize waste tobacco leaves, with significant social, economic and ecological benefits.

[0003] Tobacco proteins are divided into (water) soluble tobacco proteins and (water) insoluble tobacco proteins. About 50% of the soluble tobacco proteins are single-component chloroplast proteins (Fraction I protein, i.e. FI protein), whose full name is ribulose-1,5-bisphosphate carboxylase-oxygenase, and the rest are complexes of other soluble tobacco proteins (Fraction II protein, i.e. F-II protein). Although a lot of research has been done on soluble tobacco proteins in the industry, little research has been done on insoluble tobacco proteins, mainly due to the lack of effective preparation and purification methods.

[0004] Therefore, there is an urgent need for a method for extracting or preparing insoluble tobacco protein. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for extracting insoluble tobacco protein, wherein the insoluble tobacco protein extracted by the method has high purity, large lightness value and yellowness value, and a significant change in total color difference compared with the crude product, thereby improving the product quality of the insoluble tobacco protein. Another purpose of the present invention is to provide insoluble tobacco protein and its application.

[0006] To achieve the above object, the first aspect of the present invention relates to a method for extracting insoluble tobacco protein, comprising the following steps:

[0007] (1) mixing a crude product containing insoluble tobacco protein with chlorite in a solvent, and reacting the mixture at 15°C to 90°C (e.g., 15°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 75°C, 80°C, 90°C) to obtain a reaction product;

[0008] (2) centrifuging the reaction product and collecting the upper layer material;

[0009] (3) The upper layer material is mixed with a precipitant, and the solid-liquid separation is performed, and the solid phase is collected, which is the insoluble tobacco protein.

[0010] Without being limited by theory, impurities in the crude product containing insoluble tobacco protein form precipitates during the reaction and are removed by centrifugation; then, a precipitant is added to precipitate the insoluble tobacco protein, thereby facilitating the separation and collection of the purified insoluble tobacco protein.

[0011] In any embodiment, the mass ratio of the crude product to the chlorite is 2:1 - 25:1 or 2.5:1 - 20:1, for example 2:1, 2.5:1, 3:1, 3.3:1, 4:1, 5:1, 7:1, 9:1, 10:1, 13:1, 15:1, 18:1, 20:1, 25:1.

[0012] In any embodiment, the mass ratio of the chlorite to the precipitant is 1:48 - 1:3 or 1:32 - 1:4, for example, 1:48, 1:45, 1:40, 1:35, 1:32, 1:30, 1:25, 1:20, 1:16, 1:15, 1:10, 1:8, 1:6.67, 1:5.3, 1:5, 1:4, 1:3.

[0013] In any embodiment, in step (3), before solid-liquid separation, the mixture is allowed to stand at 0°C - 7°C (preferably 0°C - 5°C) for 8 - 14 hours (e.g. 8 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours).

[0014] In any embodiment, the chlorite is selected from one or more of sodium chlorite, potassium chlorite, magnesium chlorite, and barium chlorite, preferably sodium chlorite.

[0015] In any embodiment, the precipitant is selected from one or more ammonium salts.

[0016] In any embodiment, the precipitant is selected from one or more of ammonium sulfate, ammonium chloride, and ammonium bicarbonate, preferably ammonium sulfate.

[0017] In any embodiment, in step (1), the pH value of the chlorite is adjusted to 4-10 before mixing, or the pH value of the mixed system is adjusted to 4-10 during the mixing process, for example, the pH value is adjusted to 4, 5, 6, 7, 8, 9 or 10.

[0018] In any embodiment, in step (1), an acid solution is used to adjust the pH value. In one embodiment, the acid solution can be a glacial acetic acid solution.

[0019] In any embodiment, in step (2), the centrifugation is performed at a speed of 7000-11000 r / min, for example, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min.

[0020] In any embodiment, in step (2), the centrifugation time is 8-18 minutes, for example, 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, or 18 minutes.

[0021] In any embodiment, in step (1), the mass ratio of the chlorite to the solvent is 0.3:99.7-6:94, for example, 0.3:99.7, 0.5:99.5, 1:99, 2:98, 3:97, 4:96, 5:95, 6:94.

[0022] In any embodiment, in step (3), solid-liquid separation is performed by centrifugation.

[0023] In any embodiment, in step (3), the centrifugation is performed at a speed of 7000-11000 r / min (e.g., 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min, 11000 r / min) for 8-18 minutes (e.g., 8 minutes, 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes).

[0024] In any embodiment, the method further comprises: washing the solid phase material obtained in step (3) and freeze-drying. The freeze-drying temperature may be a conventional freeze-drying temperature. In one embodiment, the freeze-drying temperature may be -45°C to -35°C, for example -40°C.

[0025] In any embodiment, the solvent comprises water.

[0026] In any embodiment, washing is performed with water.

[0027] In the present invention, water may be production water commonly used in the art, including but not limited to deionized water, purified water, distilled water, tap water, etc.

[0028] In any embodiment, the crude product containing insoluble tobacco protein is prepared by the following steps:

[0029] 1) extracting the tobacco sample with an alkaline compound solution at 45°C - 75°C (e.g., 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C) for 0.5 - 8 hours (e.g., 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours), separating the solid from the liquid, and collecting the liquid phase;

[0030] 2) adjusting the pH value of the liquid phase to 3.5-5.5 (e.g., 3.5, 4, 4.5, 5, 5.5), separating the solid and the liquid, and collecting the solid phase;

[0031] 3) The solid phase is mixed with a solvent, the pH value of the obtained mixture is adjusted to neutral, the solid and liquid are separated, and the solid phase is collected as a crude product containing insoluble tobacco protein.

[0032] In the present invention, the pH value being neutral has the conventional meaning in the art, for example, the pH value may be 6.5-7.5.

[0033] In any embodiment, in step 2), an acid solution is used to adjust the pH value; in one embodiment, a hydrochloric acid solution is used to adjust the pH value.

[0034] In any embodiment, in step 2), before solid-liquid separation, the liquid phase after adjusting the pH value is allowed to stand at 0°C - 7°C (preferably 0°C - 5°C) for 10-14 hours (e.g. 10 hours, 11 hours, 12 hours, 13 hours, 14 hours).

[0035] In any embodiment, in step 2), solid-liquid separation is performed by filtration.

[0036] In any embodiment, in step 1), the mass ratio of the tobacco sample to the basic compound is 1:0.20 - 1:0.11, for example, 1:0.20, 1:0.18, 1:0.16, 1:0.14, 1:0.13, 1:0.128, 1:0.12, 1:0.11.

[0037] In any embodiment, in step 1), the alkaline compound is selected from one or more of alkali metal hydroxides, alkaline earth metal hydroxides, and alkali metal carbonates; optionally, the alkaline compound is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate.

[0038] In any embodiment, in step 1), extraction is carried out under stirring conditions; optionally, the stirring speed is 400-600 r / min, such as 400 r / min, 500 r / min, 600 r / min.

[0039] In any embodiment, in step 1), the concentration of the alkaline compound solution is 0.5 mass % - 1.0 mass %, for example, 0.5 mass %, 0.6 mass %, 0.8 mass %, 1.0 mass %.

[0040] In any embodiment, in step 1), before extraction, the tobacco sample is washed, dried, crushed, and sieved.

[0041] In any embodiment, in step 1), the drying process includes draining, squeezing, and drying in sequence. Optionally, the drying temperature is 60-90°C, for example 70°C.

[0042] In any embodiment, in step 1), solid-liquid separation is performed by a ceramic membrane.

[0043] In any embodiment, the step of preparing the crude product containing insoluble tobacco protein further comprises: freezing, washing, and freeze-drying the solid phase obtained in step 3). The freeze-drying temperature may be a conventional freeze-drying temperature. In one embodiment, the freeze-drying temperature may be -45°C to -35°C, for example -40°C. In some embodiments, water washing is used. The role of freezing is to facilitate washing. The freezing temperature may be a conventional freezing temperature. In one embodiment, the freezing temperature may be -25°C to -10°C, for example -20°C, -18°C, or -15°C.

[0044] In any embodiment, in step 3), the solvent comprises water.

[0045] In any embodiment, the mass ratio of the solvent in step 3) to the tobacco sample in step 1) is 1:1-5:1, such as 1:1, 2:1, 3:1, 4:1, 5:1.

[0046] In any embodiment, in step 3), an alkaline solution is used to adjust the pH value.

[0047] The second aspect of the present invention provides an insoluble tobacco protein, which is prepared by the method of the first aspect of the present invention.

[0048] In any embodiment, the purity of the insoluble tobacco protein is ≥70%, preferably ≥72%, ≥74%, ≥78%, ≥80%, ≥83%, ≥85% or ≥88%.

[0049] In any embodiment, the lightness value of the insoluble tobacco protein is 46-80, preferably 50-80, 52-80, 55-80, 60-80, 70-80 or 73-80.

[0050] In any embodiment, the yellowness value of the insoluble tobacco protein is 19-55, preferably 24-55, 27-55, 30-55, 34-55, 40-55 or 40-52.

[0051] In any embodiment, the total color difference of the insoluble tobacco protein changes from 19-65, preferably from 25-65, 30-65, 35-65, 40-65, 45-65 or 50-65.

[0052] The third aspect of the present invention relates to the use of the insoluble tobacco protein according to the second aspect of the present invention in food.

[0053] The food in the present invention has the conventional definition in the art, including but not limited to human food, animal food, adult food, children's food, elderly food, etc.

[0054] The present invention achieves one or more of the following beneficial effects:

[0055] 1. The insoluble tobacco protein extracted by the method of the present invention has high purity.

[0056] 2. The lightness and yellowness values ​​of the insoluble tobacco protein extracted by the method of the present invention are improved, and the total color difference change compared with the crude product is very obvious, indicating that the method of the present invention significantly improves the product quality of the insoluble tobacco protein. In addition, there is a positive correlation between the purity of the insoluble tobacco protein and the lightness value, yellowness value, and total color difference change value. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0058] Figure 1 It is a fitting photograph of CPE and the water-insoluble tobacco protein after purification in Examples 1-13. DETAILED DESCRIPTION

[0059] The embodiments of the present invention will be described clearly and completely below in conjunction with the examples. Obviously, the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0060] Preparation of water-insoluble tobacco protein crude extract (CPE):

[0061] Weigh 25 kg of flue-cured tobacco, wash with water, drain, squeeze, dry at 70 °C, put it in a universal grinder, and grind it through a 60-mesh sieve to obtain powdered tobacco powder (PT). Add 400 kg of 0.8 mass% NaOH aqueous solution to a 500 L reactor, preheat the reactor to 60 °C, slowly pour in 20 kg of PT, and stir at 60 °C for 2 hours at 500 r / min. After the extraction, use a ceramic membrane to filter, add 8 M HCl solution to the filtrate to adjust the pH to 4.5, and a dark brown precipitate will precipitate. Let it stand at 0 °C-5 °C for 12 hours, filter, disperse the filter residue in 50 kg of water, adjust the pH to neutral with 2 M NaOH solution, centrifuge and filter to obtain a viscous brown water-insoluble tobacco protein, freeze at -18 °C, wash with water, and freeze-dry at -40 °C to obtain a water-insoluble tobacco protein crude extract (CPE). Example 1

[0062] Add 50 g of 0.5 mass% NaClO2 solution (pH 11) to a 125 mL conical flask, adjust the pH to 9 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction is completed, put it into a centrifuge and centrifuge it at 10000 r / min for 10 min, and collect the upper layer. Add 8 g of (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0 ℃ -5 ℃ for 12 hours, centrifuge it at 10000 r / min for 10 min, collect the precipitate in the lower layer and wash it with water, and freeze-dry it at -40°C to obtain purified water-insoluble tobacco protein. Example 2

[0063] Add 50 g of 0.5 mass% NaClO2 solution (pH 11) to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction, centrifuge at 8000 r / min for 15 min to collect the upper layer. Add 8 g of (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0°C-5°C for 12 hours, centrifuge at 10000 r / min for 10 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 3

[0064] Add 50 g of 0.5 mass% NaClO2 solution (pH 11) to a 125 mL conical flask, adjust the pH to 6 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction, centrifuge at 9000 r / min for 13 min, and collect the upper layer. Add 8 g of (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0°C-5°C for 10 hours, centrifuge at 10000 r / min for 15 min, collect the precipitate in the lower layer, wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 4

[0065] Add 50 g of 0.5 mass% NaClO2 solution (pH 11) to a 125 mL conical flask, adjust the pH to 5 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction is completed, centrifuge at 9000 r / min for 10 min to collect the upper layer. Add 8 g of (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0°C-5°C for 12 hours, centrifuge at 10000 r / min for 10 min, collect the precipitate in the lower layer, wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 5

[0066] Add 50 g of 1.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction is completed, centrifuge at 8000 r / min for 15 min, and collect the upper layer. Add 8 g (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0 ℃-5 ℃ for 10 hours, centrifuge at 10000 r / min for 15 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 6

[0067] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction, centrifuge at 10000 r / min for 13 min, and collect the upper layer. Add 8 g (NH4)2SO4 to the upper layer, stir until dissolved, and let it stand at 0 ℃-5 ℃ for 10 hours. Centrifuge at 8000 r / min for 10 min, collect the precipitate in the lower layer, wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 7

[0068] Add 50 g of 4.0 mass% NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 20°C for 30 min. After the reaction is completed, centrifuge at 8000 r / min for 10 min, and collect the upper layer. Add 8 g (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0°C-5°C for 12 hours, centrifuge at 9000 r / min for 10 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 8

[0069] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 40°C for 30 min. After the reaction, centrifuge at 9000 r / min for 15 min, and collect the upper layer. Add 8 g of (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0°C-5°C for 12 hours, centrifuge at 9000 r / min for 15 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 9

[0070] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 60°C for 30 min. After the reaction is completed, centrifuge at 10000 r / min for 13 min, and collect the upper layer. Add 8 g (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0 ℃-5 ℃ for 12 hours, centrifuge at 10000 r / min for 15 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 10

[0071] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 75°C for 30 min. After the reaction, centrifuge at 8000 r / min for 15 min, and collect the upper layer. Add 8 g (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0 ℃-5 ℃ for 10 hours, centrifuge at 8000 r / min for 13 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Embodiment 11

[0072] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 60°C for 30 min. After the reaction, centrifuge at 9000 r / min for 15 min, and collect the upper layer. Add 6 g (NH4)2SO4 to the upper layer, stir until dissolved, let it stand at 0 ℃-5 ℃ for 12 hours, centrifuge at 9000 r / min for 15 min, collect the precipitate in the lower layer and wash with water, and freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Example 12

[0073] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g of CPE and stir evenly, and react at 60°C for 30 min. After the reaction, centrifuge at 9000 r / min for 13 min and collect the upper layer. Add 10 g (NH4)2SO4 to the upper layer, stir until dissolved, and let it stand at 0°C -5°C for 10 hours. Centrifuge at 10000 r / min for 13 min, collect the precipitate in the lower layer and wash with water, freeze-dry at -40°C to obtain purified water-insoluble tobacco protein. Embodiment 13

[0074] Add 50 g of 3.0 mass % NaClO2 solution to a 125 mL conical flask, adjust the pH to 8 with glacial acetic acid solution, then add 5 g CPE and stir evenly, and react at 60°C for 30 min. After the reaction, centrifuge at 8000 r / min for 10 min and collect the upper layer. Add 12 g (NH4)2SO4 to the upper layer, stir until dissolved, and let it stand at 0°C -5°C for 10 hours. Centrifuge at 10000 r / min for 10 min, collect the precipitate in the lower layer and wash with water, freeze-dry at -40°C to obtain purified water-insoluble tobacco protein.

[0075] The parameters different between Examples 2-13 and Comparative Examples 1-6 and Example 1 are shown in Table 1.

[0076] Product testing:

[0077] (1) Protein purity test

[0078] The Kjeldahl method is used for testing. The specific operations include:

[0079] a. Digestion: Accurately weigh m (g) of purified protein sample, place three parallel groups into digestion tubes, add equal amounts of digestive enzymes, and place in the digestion tubes for 40 min when the temperature of the digestion furnace reaches 420°C. The digestion solution is green and transparent. Blank test: Except that there is no protein sample in the digestion tube, the rest is the same as the above operation.

[0080] b. Distillation: After the digestate is cooled, it is moved into the reaction chamber of the distillation apparatus for distillation. The end of the condenser is inserted into a conical flask. 30 mL of 2% by mass boric acid solution is pre-added into the conical flask and 4 drops of a mixed indicator are added. Distill until the volume of the distillate is 200 mL.

[0081] c. Titration: Titrate the distillate in the conical flask with a standard HCl solution (molar concentration c is 0.1036 mol / L) until it turns light pink as the titration endpoint. Record the volume of acid solution consumed (V1) and the volume of acid solution consumed by the blank sample (V0).

[0082] The purity P (mass %) of tobacco protein was calculated according to the following formula:

[0083] P = 6.25×100%×[(V1-V0)×c×0.0140] / m

[0084] (2) Test of lightness value, yellowness value and total color difference change

[0085] The water-insoluble tobacco protein sample was evenly spread on a glass slide, and then covered with another glass slide. The colorimeter selected 5 points for each sample for measurement. The colorimeter [model CR-10, purchased from Color Spectrum Technology (Zhejiang) Co., Ltd.] used a full-band balanced LED light source for the test sample, with a wavelength interval of 10 nm and a measurement time of 1 s; the lightness value L was recorded. * , redness value a * and yellowness value b * , calculate the total color difference change ΔE according to the following formula * ab .

[0086] ΔE * ab = [(ΔL * ) 2+ (Δa * ) 2 + (Δb * )2] 1 / 2

[0087] Where, ΔE * ab is the total color difference change, ΔL * For sample L * Values ​​and control samples L * The difference in values, Δa * For sample a * Values ​​and control samples a * The difference in values, Δb * For sample b * Values ​​and control samples b * The control sample was water-insoluble crude tobacco protein extract (CPE).

[0088] According to the test results of lightness value, yellowness value and redness value, the colorimeter's own software was used to fit the photos of CPE and the water-insoluble tobacco protein after purification in Example 1-13. Figure 1 shown.

[0089] Depend on Figure 1 It can be seen that compared with CPE, the colors of the water-insoluble tobacco proteins in Examples 1-13 of the present invention after purification are all improved.

[0090] The test results are shown in Table 1-2.

[0091] Table 1 Main parameters of Examples 1-13 and Comparative Examples 1-6

[0092] “*” indicates that Comparative Example 2 used a sodium hypochlorite (NaClO) solution with a concentration of 3 mass % instead of a NaClO2 solution.

[0093] Table 2 Test results of Examples 1-13 and Comparative Examples 1-6

[0094] From Table 1-2 we can see that:

[0095] Compared with comparative example 1 in which NaClO2 is not used to react with CPE, the purity of the water-insoluble tobacco protein obtained in examples 1-13 of the present invention is significantly improved, the lightness value and the yellowness value are significantly increased, and the total color difference compared with CPE is more obvious.

[0096] Compared with the comparative example 2 using sodium hypochlorite and CPE to react, the purity of the water-insoluble tobacco protein obtained in Examples 6-7 of the present invention is significantly improved, the lightness value and the yellowness value are significantly increased, and the total color difference compared with CPE is more obvious. The purity of the water-insoluble tobacco protein obtained in Example 5 of the present invention is significantly improved, the lightness value is significantly increased, and the total color difference compared with CPE is more obvious. The purity of the water-insoluble tobacco protein obtained in Example 2 of the present invention is significantly improved, and the yellowness value is significantly increased.

[0097] Compared with the comparative example 3 in which the weight ratio of NaClO2 to (NH4)2SO4 is too low, the purity of the water-insoluble tobacco protein obtained in Examples 2 and 5-7 of the present invention is significantly improved, the lightness value and yellowness value are significantly increased, and the total color difference compared with CPE is more obvious.

[0098] Compared with comparative example 4 in which the weight ratio of NaClO2 to (NH4)2SO4 is too high, the purity of the water-insoluble tobacco protein obtained in Examples 2, 5-7 of the present invention is significantly improved, the lightness value and yellowness value are significantly increased, and the total color difference compared with CPE is more obvious.

[0099] Compared with the reaction temperature of comparative example 5 being too high, the purity of the water-insoluble tobacco protein obtained in examples 6, 8-10 of the present invention is significantly improved, the lightness value and the yellowness value are significantly increased, and the total color difference compared with CPE is more obvious.

[0100] The pH values ​​of the reaction systems of Examples 1-4 are different. Among them, when the pH value of the reaction system of Example 2 is 8, the water-insoluble tobacco protein obtained has the highest purity, the largest lightness value and the largest yellowness value, and the most obvious total color difference compared with CPE.

[0101] The reaction temperatures of Example 2 and Examples 8-10 are different. Among them, the water-insoluble tobacco protein obtained when the reaction temperature of Example 9 is 60°C has the highest purity, the largest lightness value and yellowness value, and the most obvious total color difference compared with CPE.

[0102] The weight ratios of NaClO2 and (NH4)2SO4 in Example 9 and Examples 11-13 are different. Among them, the water-insoluble tobacco protein obtained when the weight ratio of NaClO2 and (NH4)2SO4 in Example 12 is 1:6.67 has the highest purity, the largest lightness value and yellowness value, and the most obvious total color difference compared with CPE.

[0103] The weight ratios of NaClO2 and (NH4)2SO4 in Example 2 and Examples 5-7 are different. Among them, the water-insoluble tobacco protein obtained when the weight ratio of NaClO2 and (NH4)2SO4 in Example 6 is 1:5.3 has the highest purity, the largest lightness value and yellowness value, and the most obvious total color difference compared with CPE.

[0104] Among Examples 1-13, the water-insoluble tobacco protein obtained in Example 12 has the highest purity, the largest lightness value and yellowness value, and the most obvious total color difference change compared with CPE. In addition, there is a positive correlation between the purity value of the water-insoluble tobacco protein and the lightness value, yellowness value and the total color difference change value.

[0105] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for extracting insoluble tobacco protein, comprising the following steps: (1) mixing a crude product containing insoluble tobacco protein and chlorite in a solvent, reacting at 15°C to 90°C to obtain a reaction product; (2) centrifuging the reaction product and collecting the upper layer material; (3) The upper layer material is mixed with a precipitant, and the solid-liquid separation is performed, and the solid phase is collected, which is the insoluble tobacco protein.

2. The method according to claim 1, wherein: The mass ratio of the crude product to the chlorite is 2:1-25:

1.

3. The method according to claim 1 or 2, wherein: The mass ratio of the chlorite to the precipitant is 1:48-1:

3.

4. The method according to any one of claims 1 to 3, wherein: In step (3), the mixture is allowed to stand at 0°C - 7°C for 8 - 14 hours before solid-liquid separation.

5. The method according to any one of claims 1 to 4, wherein: The chlorite is selected from one or more of sodium chlorite, potassium chlorite, magnesium chlorite and barium chlorite, preferably sodium chlorite; and / or, The precipitant is selected from one or more of ammonium salts; and / or, The precipitant is selected from one or more of ammonium sulfate, ammonium chloride, and ammonium bicarbonate, preferably ammonium sulfate.

6. The method according to any one of claims 1 to 5, comprising one or more of the following: A) in step (1), the pH value of the chlorite is adjusted to 4-10 before mixing, or the pH value of the mixed system is adjusted to 4-10 during the mixing process; B) In step (2), centrifuging at 7000-11000 r / min; C) In step (2), the centrifugation time is 8-18 minutes; D) in step (1), the mass ratio of the chlorite to the solvent is 0.3:99.7 - 6:94; E) in step (3), solid-liquid separation is performed by centrifugation; F) In step (3), centrifuging at 7000-11000 r / min for 8-18 minutes; G) The method further comprises: washing and freeze-drying the solid phase obtained in step (3); H) The solvent comprises water.

7. The method according to any one of claims 1 to 6, wherein: The crude product containing insoluble tobacco protein is prepared by the following steps: 1) Extracting tobacco samples with an alkaline compound solution at 45°C - 75°C for 0.5 - 8 hours, separating the solid and the liquid, and collecting the liquid phase; 2) adjusting the pH value of the liquid phase to 3.5-5.5, separating the solid and the liquid, and collecting the solid phase; 3) The solid phase is mixed with a solvent, the pH value of the obtained mixture is adjusted to neutral, the solid and liquid are separated, and the solid phase is collected as a crude product containing insoluble tobacco protein.

8. The method according to claim 7, wherein: The steps of preparing the crude product containing insoluble tobacco protein may further include one or more of the following: a) In step 2), before solid-liquid separation, the liquid phase after adjusting the pH value is allowed to stand at 0°C - 7°C for 10-14 hours; b) In step 1), the mass ratio of the tobacco sample to the alkaline compound is 1:0.2 - 1:0.11, c) In step 1), the alkaline compound is selected from one or more of alkali metal hydroxides, alkaline earth metal hydroxides, and alkali metal carbonates; alternatively, the alkaline compound is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate; d) in step 1), extracting under stirring; optionally, the stirring speed is 400-600 r / min; e) In step 1), the concentration of the alkaline compound solution is 0.5% by mass - 1.0% by mass; f) in step 1), before extraction, the tobacco sample is washed, dried, crushed and sieved; g) further comprising: freezing, washing and freeze-drying the solid phase obtained in step 3).

9. An insoluble tobacco protein obtained by the method according to any one of claims 1 to 8; Optionally, the insoluble tobacco protein comprises one or more of the following: The purity of the insoluble tobacco protein is ≥70%; The lightness value of the insoluble tobacco protein is 46-80; The yellowness value of the insoluble tobacco protein is 19-55; The total color difference of the insoluble tobacco protein varies from 19-65.

10. Use of the insoluble tobacco protein according to claim 9 in food.

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