Camellia oleifera protein and tea saponin prepared synchronously as well as preparation method and application of camellia oleifera protein and tea saponin

Abstract

The invention belongs to the technical field of comprehensive utilization of oil cakes, and discloses camellia oleifera protein and tea saponin which are prepared synchronously, and a preparation method and application thereof. Aiming at the problems of complex extraction process steps, low efficiency and poor edibility caused by saponin accompanying in the oil tea protein extraction process, the invention adopts a weak acid high-salt dissolution-ethanol low-temperature precipitation separation combined technology, and comprises the following steps: firstly, performing weak acid high-salt treatment on degreased oil tea meal to enrich protein; the combination of substances such as saponin and polyphenol with protein is weakened; then adding precooled ethanol to dehydrate and precipitate the protein, and keeping good solubility of saponin in the ethanol supernatant; according to the method, removal of high salt and enrichment and purification of oil-tea camellia protein can be synchronously realized through a coupling technique, and the low-saponin and high-purity oil-tea camellia protein can be obtained by directly drying the precipitate obtained after low-temperature precipitation separation of ethanol at room temperature; the supernate is a tea saponin concentrated solution, so that co-production of the oil tea protein and the tea saponin is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of comprehensive utilization of oilseed cakes and meals, and particularly relates to camellia protein and tea saponin prepared synchronously, and a preparation method and application thereof. Background Art

[0002] Camellia is one of the four major woody oil crops. Its protein has not been fully utilized like soybean protein. The key limiting step lies in the coexistence of a large amount of saponin, resulting in a pungent taste and poor edibility. Camellia saponin has various functions such as antibacterial, antioxidant, anti-inflammatory, and improving protein functional properties, and is widely used in beauty and health care skin care products, but it also has certain hemolytic and gastrointestinal toxicity. Therefore, camellia saponin is a double-edged sword for camellia protein. Whether it can efficiently achieve the combined extraction and separation of the two urgently needs to be broken through.

[0003] The existing extraction methods of camellia protein mainly include alkali extraction-acid precipitation method, enzymatic method, Osborne fractionation method, three-phase extraction technology, reverse micelle extraction method, etc.

[0004] The alkali extraction-acid precipitation method is one of the most traditional protein extraction methods. The process flow is simple, the production cost is low, and the extraction rate is high. However, excessive alkali is likely to cause protein denaturation. Some studies combine physical methods such as ultrasonic and steam explosion with the alkali extraction method to improve the protein dissolution efficiency. For example, Zhou Yibin et al. used ultrasonic-assisted extraction of protein from camellia meal. The optimal extraction conditions were pH 9.0, ultrasonic power 350W, ultrasonic time 35min, and solid-liquid ratio 1:3 (m / v), and the protein extraction rate reached 70.1%. Zhang Shanying et al. used steam explosion-assisted extraction. The optimal extraction conditions were solid-liquid ratio 1:10, extraction temperature 40°C, pH 10, and extraction time 50min, and the protein extraction rate was 71.0%.

[0005] The salt method is simple to operate, but the extraction time is long, an additional desalting process is required, and the purification multiple is not high.

[0006] The enzymatic method requires the addition of amylase, cellulase, and alkaline protease for catalysis, and the enzymatic hydrolysis temperature is controlled at 45-70°C for 30-120min. The protein extraction rate can reach 80%. The enzymatic method for extracting protein requires a short time and high efficiency, but has high requirements for external extraction conditions. Inappropriate conditions will cause the loss of enzyme activity and reduce the protein extraction efficiency.

[0007] The protein resources in camellia cake and meal have not been well developed and utilized. In addition to the above problems, there is also a very important point: the content of saponin in camellia is extremely high, up to 30% of the dry weight of camellia meal. It is co-dissolved, enriched, and precipitated with protein during the extraction process, making it difficult to improve the purity of protein. In addition, camellia saponin has hemolytic toxicity and a bitter, pungent taste, and its coexistence with camellia protein will greatly limit its application in the fields of feed, food, etc.

[0008] Some studies also pre-treat camellia seed meal with organic solvents (such as ethanol, isopropanol, etc.) before extraction to remove some saponins in advance. For example, before extracting proteins, Liu Jing et al. used 75% ethanol to extract 5 times, 1 hour each time, to remove substances such as saponins and polyphenols in camellia seed meal. However, this not only increases the process burden but also denatures the proteins, resulting in a protein extraction rate of only 41.9%.

[0009] Therefore, it is of great significance to develop a preparation method that can take into account the extraction cost and nutritional utility, and at the same time realize the synchronous extraction of camellia protein and tea saponin. Summary of the Invention

[0010] In view of this, the present invention adopts the weak acid high-salt dissolution combined with ethanol low-temperature precipitation separation technology, that is, first uses weak acid high-salt to enrich proteins and tea saponins in camellia seed meal, and then uses ethanol low-temperature precipitation separation technology to synchronously separate proteins and tea saponins, achieving desalination, concentration, enrichment and separation of camellia protein and tea saponin in one step, and finally obtaining a crude extract of camellia protein and tea saponin with low saponin and high purity.

[0011] In order to achieve the above object, the first object of the present invention is to provide a synchronous preparation method of camellia protein and tea saponin, adopting the following technical scheme:

[0012] A synchronous preparation method of camellia protein and tea saponin, using the weak acid high-salt dissolution - ethanol low-temperature precipitation separation combined technology to synchronously prepare a concentrated solution of camellia protein and tea saponin.

[0013] It is worth noting that although there have been reports on the use of weak acid high-salt dissolution or ethanol low-temperature precipitation separation alone to prepare proteins from other sources in the prior art, there is still a technical blank in the combined use of the two, especially in the field of synchronous extraction and preparation of camellia protein and tea saponin.

[0014] On the one hand, the weak acid high-salt technology of the present invention can fully dissolve albumin and globulin in camellia protein; on the other hand, there is impurity competition inhibition, that is, salt ions preferentially bind to polyphenols and saponins in camellia seed meal, reducing their binding to camellia protein, which is beneficial to the subsequent efficient separation of the two by combining ethanol low-temperature precipitation separation. The ethanol low-temperature precipitation separation technology takes into account that polyphenols and saponins have high solubility in ethanol, and the pH in the weak acid range is close to the isoelectric point of camellia protein. After adding ethanol, the solubility of camellia protein drops sharply, resulting in the aggregation and precipitation of protein molecules. Therefore, based on the difference in solubility and the competition inhibition effect, the efficient separation of camellia protein and tea saponin can be achieved.

[0015] In addition, the traditional method for removing co-extracted polyphenols and small molecule co-existing substances from plant proteins is based on methods such as membrane filtration, osmosis, or nanofiltration, which separate based on molecular weight differences. However, the co-extract obtained by dissolving camellia oleifera protein in weak acid and high salt contains a large amount of saponins. Saponins have strong foaming properties. Even a small amount of dissolved air in methods such as membrane filtration, osmosis, or nanofiltration can generate a large number of bubbles, resulting in the inability to carry out separation. Therefore, in view of the problems of high saponin content and strong foaming properties during the extraction process of camellia oleifera protein, the present invention adopts a combined technology of weak acid high salt dissolution - ethanol low temperature precipitation separation, which can not only increase the dissolution of camellia oleifera protein, reduce the binding of camellia oleifera protein to saponins and polyphenols, but also efficiently separate polyphenols, saponins and camellia oleifera protein. In particular, after efficiently removing polyphenols, the color of camellia oleifera protein powder has been improved, and the whiteness value has increased, which is more conducive to the market and promotion of products.

[0016] Furthermore, the specific steps of the combined technology of weak acid high salt dissolution - ethanol low temperature precipitation separation include:

[0017] (1) Camellia oleifera meal is mixed with a salt solution with an ionic strength of 0.5 - 2.0 mol / L at a solid-liquid ratio of 1:5 - 1:30 (m / v), stirred and dissolved at pH 6.0, and the solid-liquid separation is carried out to retain the camellia oleifera protein extract;

[0018] (2) The protein concentration of the protein extract obtained in step (1) is controlled to be 0.5 - 4.0 mg / mL using ultrapure water, and then pre-cooled absolute ethanol is gradually added, and the solid-liquid separation is carried out to obtain the supernatant and the precipitate. The precipitate is dried at room temperature to obtain the low-saponin camellia oleifera protein, and the supernatant is the concentrated solution of tea saponin.

[0019] It should be noted that camellia seed cake is rich in protein and saponin, both of which have amphiphilic properties and the solubility decreases with the decrease of the pH value of the aqueous solution. Similar physicochemical properties make the separation of the two extremely difficult. In order to improve the final yield of the crude protein and saponin extracts, the present invention selects weak acid and high salt conditions. At the same time, in order to efficiently, greenly and industrially separate camellia protein and tea saponin, ethanol low-temperature precipitation separation is selected. During the process of promoting dissolution by weak acid and high salt extraction, high-concentration salt ions promote protein dissolution and inhibit the interaction between proteins and small molecules such as saponin and polyphenol. And in a weak acid environment, small molecules with antioxidant properties are not easily oxidized and are not easily covalently bound to proteins, which is more conducive to the subsequent separation of tea saponin and protein. On the contrary, alkaline conditions will cause polyphenol small molecules to be easily oxidized and covalently interact with proteins, which will not only greatly increase the separation difficulty, but also easily lead to the poor color of the protein. During the process of ethanol low-temperature precipitation separation, ethanol can effectively destroy the hydration layer of proteins, so that both albumin and globulin in camellia protein can precipitate rapidly, and low temperature will be beneficial to protecting proteins and reducing their denaturation degree. In addition, saponin and polyphenol have extremely high solubility in ethanol. Therefore, ethanol low-temperature precipitation separation can effectively separate non-covalently bound saponin from camellia protein to obtain camellia protein with low saponin and high purity and tea saponin concentrate.

[0020] Specifically, the present invention adopts a combined technology of weak acid and high salt dissolution - ethanol low-temperature precipitation separation. Among them, the weak acid and high salt conditions are beneficial to enhancing the subsequent ethanol low-temperature precipitation separation effect, because under weak acid conditions, proteins are close to their isoelectric points and have low hydration degree, which is very conducive to further dehydration and precipitation by ethanol.

[0021] Furthermore, in the step (1), the salt solution includes one of NaCl, KCl, CaCl2, and MgCl2 salt solutions.

[0022] Furthermore, in the step (2), absolute ethanol is pre-cooled to ≤ -20 °C, and the volume ratio of the protein extract to ethanol is controlled to be 5:2 to 5:3.

[0023] Furthermore, the solid-liquid separation methods in the steps (1) and (2) include centrifugal separation or filtration separation.

[0024] Preferably, the solid-liquid separation method is centrifugal separation.

[0025] In some embodiments, the solid-liquid separation method is centrifugation at 15000g for 20 min at 4 °C.

[0026] The second object of the present invention is to provide a camellia protein and tea saponin concentrate prepared by the preparation method as described above.

[0027] In addition, the third object of the present invention is to provide the application of the camellia protein in the fields of feed and food processing, and the application of the tea saponin concentrate in the fields of daily chemical products and medicine.

[0028] It should be noted that the low-saponin camellia protein provided by the present invention, as a plant-derived protein, can be used as an alternative protein in fields such as food emulsification, foaming, and film-forming; it can also be used as a new resource food ingredient to enrich the sources of food raw materials.

[0029] Compared with the prior art, in view of the problems of complex extraction process steps, low efficiency, and poor edibility caused by the accompanying saponin during the extraction process of camellia protein, the present invention adopts a combined technology of weak acid high-salt dissolution - ethanol low-temperature precipitation separation, that is, after degreasing the camellia meal, the protein is first enriched by weak acid high-salt treatment, and the binding of substances such as saponin and polyphenol to the protein is weakened. Then, pre-cooled ethanol is added to dehydrate and precipitate the protein, while the saponin still maintains good solubility in the ethanol supernatant. Through this combined technology, high-salt removal, concentration, and enrichment of camellia protein are achieved in one step, and low-saponin and high-purity camellia protein can be directly obtained by drying; in addition, the supernatant obtained after ethanol cold precipitation treatment is the tea saponin concentrate, and this technology realizes the co-production of camellia protein and tea saponin. The preparation method of the present invention is simple, the process is green and environmentally friendly, and it is easy to realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0031] Figure 1 It is a process flow chart of the preparation method of the low-saponin camellia protein disclosed by the present invention.

[0032] Figure 2 It is the protein recovery rate of camellia protein obtained by different extraction methods disclosed in Examples 1 - 8 and Comparative Examples 1 - 2 of the present invention.

[0033] Figure 3 It is the saponin / protein ratio of camellia protein obtained by different extraction methods disclosed in Examples 1 - 8 and Comparative Examples 1 - 2 of the present invention.

[0034] Figure 4 It is the appearance pictures of camellia protein obtained by different extraction methods disclosed in Examples 1 - 8 and Comparative Examples 1 - 2 of the present invention.

[0035] Figure 5The whiteness values of camellia protein obtained by different extraction methods disclosed in Examples 1-8 and Comparative Examples 1-2 of the present invention. Detailed implementation manners

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] The special term "embodiment" used here, any embodiment described as "exemplary" does not have to be construed as superior or better than other embodiments. For the performance index tests in the embodiments of the present application, unless otherwise specified, the conventional test methods in the art are adopted. It should be understood that the terms described in the present application are only for describing specific embodiments and are not used to limit the content disclosed in the present application.

[0038] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present application belongs; the test methods and technical means not specifically mentioned in the present application are all the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0039] To better illustrate the content of the present application, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present application can also be implemented without some specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present application.

[0040] On the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of the present application.

[0041] The present invention discloses a method for synchronously preparing camellia protein and tea saponin, and the preparation method and application thereof, belonging to the technical field of comprehensive utilization of oilseed cakes. Aiming at the problems of complex extraction process steps, low efficiency and poor edibility caused by the accompanying saponin during the extraction process of camellia protein, the present invention adopts a combined technology of weak acid and high salt dissolution - ethanol low temperature precipitation separation, that is, after defatting the camellia meal, the protein is first enriched by weak acid and high salt treatment, and the binding of substances such as saponin and polyphenol to the protein is weakened. Then, pre-cooled ethanol is added to dehydrate and precipitate the protein, and the saponin still maintains good solubility in the ethanol supernatant. Through this combined technology, high salt is removed and camellia protein is concentrated and enriched in one step, and low-saponin and high-purity camellia protein can be directly obtained by drying; in addition, the supernatant obtained after ethanol cold precipitation treatment is the tea saponin concentrate, and this technology realizes the co-production of camellia protein and tea saponin. The preparation method of the present invention is simple, the process is green and environmentally friendly, and it is easy to realize industrial mass production.

[0042] To better understand the present invention, the following examples are used to further specifically illustrate the present invention, but it should not be understood as a limitation to the present invention. For some non-essential improvements and adjustments made by those skilled in the art according to the above-mentioned invention content, they are also considered to fall within the protection scope of the present invention.

[0043] The experiments and characterization methods involved in the present invention:

[0044] 1. Determination of protein concentration:

[0045] The BCA method is used to determine the protein concentration. Take 20 μL of bovine serum albumin standard solution with a protein concentration of 0 - 1.0 mg / mL and add it to a 96-well plate. Add 200 μL of BCA working solution to each well, and measure the absorbance at 562 nm. The standard curve is Y = 0.6546X + 0.1382, R 2 = 0.999, where: X represents the protein mass concentration / (mg / mL), and Y represents the absorbance. Determine the protein content in the sample. Dissolve the protein precipitate in 0.5% SDS. After full dissolution, take 20 μL of the sample, add 200 μL of BCA working solution according to the above method, measure the absorbance value at 562 nm, and calculate the protein concentration according to the standard curve.

[0046] 2. Determination of saponin concentration:

[0047] The vanillin - sulfuric acid method was used to determine the content of tea saponin. Weigh the saponin standard product and dissolve it in 80% ethanol solution to prepare a standard solution with a concentration range of 0 - 1.5 mg / mL. Take 0.5 mL and put it into a stoppered test tube. Under an ice - water bath, successively add 0.5 mL of 8% vanillin - ethanol solution and 4 mL of 77% sulfuric acid solution. After mixing, react in a water bath at 60 °C for 20 min. After the reaction, cool it in an ice - water bath for 10 min, place it at room temperature for 10 min, and then measure the absorbance at 545 nm. The standard curve is Y = 0.2531X + 0.2464, R 2 = 0.9917, where: X represents the saponin mass concentration / (mg / mL), and Y represents the absorbance. To determine the content of tea saponin in the sample, take 0.5 mL of the sample, add vanillin - ethanol and concentrated sulfuric acid successively according to the above method for reaction, measure the absorbance at 545 nm, and calculate the saponin concentration based on the standard curve.

[0048] 3. Calculation of protein recovery rate:

[0049] Protein recovery rate = Precipitated protein content / Protein content in the extract

[0050] 4. Calculation of saponin / protein ratio:

[0051] Saponin / protein ratio = Saponin concentration / Precipitated protein content

[0052] Example 1

[0053] Take camellia seed meal, mix it with an NaCl salt solution with an ionic strength of 0.5 mol / L according to a solid - liquid ratio of 1:20 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 0.5 mg / mL, and drop - wise add anhydrous ethanol precooled at - 20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain the supernatant and precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin. Among them, the proportion of saponin in the concentrated solution in the total saponin is 88.3%.

[0054] Example 2

[0055] Take camellia seed meal, mix it with a NaCl salt solution with an ionic strength of 0.5 mol / L according to a solid-liquid ratio of 1:20 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin. Among them, the proportion of saponin in the concentrated solution accounts for 93.3% of the total saponin.

[0056] Example 3

[0057] Take camellia seed meal, mix it with a NaCl salt solution with an ionic strength of 0.5 mol / L according to a solid-liquid ratio of 1:20 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 4.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin. Among them, the proportion of saponin in the concentrated solution accounts for 84.2% of the total saponin.

[0058] Example 4

[0059] Take camellia seed meal, mix it with a NaCl salt solution with an ionic strength of 1.0 mol / L according to a solid-liquid ratio of 1:10 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:3. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin.

[0060] Example 5

[0061] Take camellia seed meal, mix it with a NaCl salt solution with an ionic strength of 2.0 mol / L according to a solid-liquid ratio of 1:10 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:3. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin.

[0062] Example 6

[0063] Take camellia seed meal, mix it with a KCl salt solution with an ionic strength of 2.0 mol / L according to a solid-liquid ratio of 1:20 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin.

[0064] Example 7

[0065] Take camellia seed meal, mix it with a CaCl2 salt solution with an ionic strength of 1.0 mol / L according to a solid-liquid ratio of 1:10 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin.

[0066] Example 8

[0067] Take camellia seed meal, mix it with a MgCl2 salt solution with an ionic strength of 1.0 mol / L at a solid-liquid ratio of 1:15 (m / v), adjust the pH to 6.0, stir and dissolve for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a supernatant and a precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrated solution of tea saponin.

[0068] To further prove the beneficial effects of the present invention for better understanding of the present invention, the following comparative examples further illustrate the properties and application performance of the simultaneously prepared camellia protein and tea saponin and their preparation methods disclosed by the present invention. However, it should not be construed as a limitation of the present invention. For those skilled in the art, the method properties obtained from other determination experiments based on the above invention content and the applications based on the above properties are also considered to fall within the protection scope of the present invention.

[0069] Comparative Example 1

[0070] Take camellia seed meal, mix it with ultrapure water at a solid-liquid ratio of 1:20 (m / v), adjust the pH to 6.0, stir and extract for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract. Dilute the camellia protein extract with ultrapure water to a protein concentration of 2.0 mg / mL, and gradually add anhydrous ethanol pre-cooled at -20 °C for 12 h until the volume ratio of the protein extract to ethanol is 5:2. Centrifuge (15000 g, 20 min, 4 °C) to obtain a precipitate, and dry it at room temperature to obtain camellia protein.

[0071] Comparative Example 2

[0072] Take camellia seed meal, mix it with ultrapure water at a solid-liquid ratio of 1:10 (m / v), adjust the pH to 8.0, stir and extract for 1 h, centrifuge at 15000 g and 4 °C for 20 min to obtain a camellia protein extract, then adjust the protein extract to pH 3.5, centrifuge (15000 g, 20 min, 4 °C) to obtain a precipitate, and finally freeze-dry to obtain camellia protein.

[0073] The core parameters of Comparative Examples 1-2 and Examples 1-8 are shown in Table 1.

[0074] Table 1

[0075]

[0076]

[0077] Through characterization and experimental comparison, Figure 2 it can be seen that the protein recovery rates of all samples in the examples are higher than those in the comparative examples. Comparing Examples 1-8 with Comparative Example 1 shows that weak acid and high salt dissolution is beneficial to a significant improvement in protein recovery efficiency; comparing Examples 1-8 with Comparative Example 2 shows that ethanol low-temperature precipitation separation has a better protein recovery rate than the conventional alkali dissolution and acid precipitation method. The high protein recovery rates of Examples 1-8 are affected by both the promotion of protein dissolution by high-concentration salt ions during the extraction process and the fact that in a weak acid environment, the protein is close to its isoelectric point, and ethanol can more effectively destroy the hydration layer of the protein, enabling both albumin and globulin in camellia protein to precipitate rapidly, and low temperature will protect the protein and reduce its denaturation degree.

[0078] Figure 3 Among them, the saponin / protein ratios of all samples in the examples are lower than those in the comparative examples. Comparing Examples 1-8 with Comparative Example 1 shows that weak acid and high salt dissolution is beneficial to removing saponin and obtaining higher-purity protein; comparing Examples 1-8 with Comparative Example 2 shows that ethanol low-temperature precipitation separation is more conducive to removing saponin and obtaining higher-purity protein than the conventional acid precipitation method. The low saponin / protein ratios of Examples 1-8 are due, on the one hand, to the inhibition of the binding between protein and saponin under the weak acid and high salt extraction conditions, and on the other hand, the extremely high solubility of saponin in ethanol, which can effectively separate the non-covalently bound saponin from camellia protein to obtain high-purity camellia protein.

[0079] In terms of color and appearance, Figure 4 and Figure 5 among them, the whiteness of the samples in the examples is higher than that in Comparative Example 1, indicating that the use of high-concentration salt ions inhibits the interaction between proteins and small molecules such as polyphenols, and in a weak acid environment, small antioxidant substances are not easily oxidized, and the separation of polyphenols, saponin and protein is promoted during the ethanol low-temperature precipitation separation process, improving the whiteness; Examples 7 and 8 obtained higher whiteness, indicating that Ca 2+ and Mg 2+ have a better effect of inhibiting the binding of polyphenols or pigments to proteins.

[0080] In summary, the preparation method of low-saponin camellia protein disclosed in the present invention aims at the problems in the extraction process of camellia protein, such as the complexity of the extraction process steps, low efficiency and poor edibility caused by the accompanying saponin. By using weak acid and high salt dissolution and combining ethanol low-temperature precipitation separation technology, it realizes the removal of high salt and the concentration and enrichment of camellia protein in one step, and directly drying can obtain low-saponin and high-purity camellia protein.

[0081] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A synchronous preparation method of camellia protein and tea saponin, characterized in that The camellia protein and saponin concentrate are simultaneously prepared by using the combined technology of weak acid high-salt dissolution - low-temperature ethanol precipitation separation.

2. The preparation method according to claim 1, characterized in that, The specific steps of the combined technology of weak acid high-salt dissolution - low-temperature ethanol precipitation separation include: (1) Camellia meal is mixed with a salt solution with an ionic strength of 0.5 - 2.0 mol / L at a solid-to-liquid ratio of 1:5 - 1:30 (m / v), stirred and dissolved under the condition of pH 6.0, and the solid-liquid separation is carried out to retain the camellia protein extract; (2) The protein concentration of the protein extract obtained in step (1) is controlled to be 0.5 - 4.0 mg / mL by using ultrapure water, and then pre-cooled absolute ethanol is gradually added, and solid-liquid separation is carried out to obtain the supernatant and the precipitate. The precipitate is dried at room temperature to obtain the camellia protein, and the supernatant is the concentrate of saponin.

3. The preparation method according to claim 2, characterized in that, In step (1), the salt solution includes one of NaCl, KCl, CaCl2, and MgCl2 salt solutions.

4. The preparation method according to claim 2, characterized in that, In step (2), the absolute ethanol is pre-cooled to ≤ -20 °C, and the volume ratio of the protein extract to ethanol is controlled to be 5:2 - 5:

3.

5. The preparation method according to any one of claims 3 or 4, characterized in that, The methods of solid-liquid separation in steps (1) and (2) include centrifugal separation or filtration separation.

6. The camellia protein and saponin concentrate prepared by the preparation method according to any one of claims 1 - 5.

7. The application of the camellia protein according to claim 6 in the fields of feed and food processing.

8. The application of the saponin concentrate according to claim 6 in the fields of daily chemical products and medicine.

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