A method for extracellular preparation of covalently grafted product of rice bran protein and Phyllanthus emblica polysaccharide

Abstract

The present invention discloses a method for extracellular preparation of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide, which relates to the field of biotechnology. The method comprises the following steps: mixing fresh Phyllanthus emblica fruits, rice bran protein and water, and then performing extraction treatment with a flash extractor under ultrasonic assistance, followed by filtration to obtain a filtrate, concentrating the filtrate to obtain a concentrated solution, then obtaining a precipitate through alcohol precipitation, and freeze - drying to obtain the covalently grafted rice bran protein - Phyllanthus emblica polysaccharide. By combining the maximum rotational speed shearing of the flash extractor and ultrasonic assistance, this method realizes the one - step high - efficiency extraction of Phyllanthus emblica polysaccharide, and by controlling the Maillard reaction conditions, a covalently grafted rice bran protein - Phyllanthus emblica polysaccharide is formed while extracting the polysaccharide, which not only significantly improves the yield and purity of the grafted product, but also ensures the maximization of the stability and functionality of the product structure.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for extracellular preparation of covalently grafted rice bran protein-phyllanthus emblica polysaccharide conjugates. Background Art

[0002] Phyllanthus emblica polysaccharide (PEP) is a natural polysaccharide extracted from Phyllanthus emblica. Polysaccharides are macromolecular polymers formed by the connection of multiple monosaccharide molecules through glycosidic bonds. Rice bran protein (RBP) is a high-quality plant protein with good solubility and foaming properties. Rice bran protein can also be used as a food additive to improve the texture and taste of food. The present invention intends to combine the two through covalent grafting technology to develop a new type of food additive, thereby improving the texture of food and increasing the water retention and nutritional value of the product.

[0003] In the preparation of protein and polysaccharide polymers, there are chemical reagent cross-linking methods, enzyme cross-linking methods, and Maillard reaction methods. The chemical reagent cross-linking method has the advantages of rapid reaction and high cross-linking efficiency. However, the cross-linking reagent itself has certain chemical toxicity and will cause irritating reactions to the human body, and the chemical reagent cross-linking method is limited to certain types of sugars, and sugars without free amino groups cannot form intermolecular covalent cross-links with proteins; the enzyme cross-linking method has the characteristics of being green, non-toxic, and having strong reaction specificity. However, its reaction efficiency is not high, the cost is high, and the restriction on the substrate is strong. Many sugars without phenolic acid structures and amino groups cannot be used to prepare covalent complexes with proteins by enzyme cross-linking. Summary of the Invention

[0004] The object of the present invention is to provide a method for extracellular preparation of covalently grafted rice bran protein-phyllanthus emblica polysaccharide conjugates to solve the problems existing in the above-mentioned prior art. This method realizes the one-step high-efficiency extraction of phyllanthus emblica polysaccharide by combining the maximum rotational speed shearing of a flash extractor and ultrasonic assistance, and forms covalently grafted rice bran protein-phyllanthus emblica polysaccharide conjugates (RBP-PEP) while extracting polysaccharides by controlling the Maillard reaction conditions. This method can rapidly dissolve the intracellular polysaccharides of phyllanthus emblica, realize the high-efficiency covalent grafting between rice bran protein and phyllanthus emblica polysaccharide, not only significantly improve the yield and purity of the grafted product, but also ensure the maximization of the stability and functionality of the product structure.

[0005] To achieve the above object, the present invention provides the following solution:

[0006] The present invention provides a method for extracellular preparation of covalently grafted rice bran protein-phyllanthus emblica polysaccharide conjugates, comprising the following steps:

[0007] The fresh Phyllanthus emblica fruits, rice bran protein and water are mixed, and then under the assistance of ultrasound, extraction treatment is carried out using a flash extractor. After that, filtration is performed to obtain a filtrate, the filtrate is concentrated to obtain a concentrated solution, and then precipitation is obtained through alcohol precipitation treatment. After freeze-drying, the rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate is obtained.

[0008] Further, the ratio of the total mass of the fresh Phyllanthus emblica fruits and the rice bran protein to the mass of the water is 1:35.

[0009] Further, the pH of the reaction system composed of the fresh Phyllanthus emblica fruits, the rice bran protein and the water is 6.5.

[0010] Further, the concentration treatment is to concentrate the filtrate to 1 / 4 - 1 / 5 of the original volume.

[0011] Further, the alcohol precipitation treatment is to add ethanol to the concentrated solution to make its volume fraction 90%.

[0012] Further, the mass ratio of the fresh Phyllanthus emblica fruits to the rice bran protein is 1:5; and / or

[0013] the ultrasonic power of the ultrasonic assistance is 640 W; and / or

[0014] the extraction temperature of the extraction treatment is 70 °C; and / or

[0015] the extraction time of the extraction treatment is 200 s.

[0016] The present invention also provides a rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate prepared by the method according to the above.

[0017] The present invention also provides the application of the above rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate in the preparation of food additives.

[0018] The present invention also provides a food additive comprising the above rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate.

[0019] The present invention also provides the application of the above food additive in the preparation of food.

[0020] The present invention discloses the following technical effects:

[0021] The present invention develops a method for extracellular preparation of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide. By combining the maximum rotational speed shearing of a flash extractor and ultrasonic assistance, efficient extraction of Phyllanthus emblica polysaccharide in one step is achieved. And by controlling the Maillard reaction conditions, covalently grafted rice bran protein - Phyllanthus emblica polysaccharide (RBP - PEP) is formed while extracting the polysaccharide, which is an innovation compared to traditional extraction and grafting methods that require multiple steps. This method can rapidly dissolve intracellular polysaccharide of Phyllanthus emblica, achieve high - efficiency covalent grafting between rice bran protein and Phyllanthus emblica polysaccharide, not only significantly improving the yield and purity of the grafted product, but also ensuring the maximization of the stability and functionality of the product structure.

[0022] In the method of the present invention, the mechanical shearing force and cavitation effect generated by the ultrasonic - assisted flash evaporator can break the plant cell wall, enhance the binding of polysaccharide and protein, and improve the grafting efficiency; the high - frequency vibration of ultrasonic waves promotes solvent penetration and polysaccharide dissolution, realizing the rapid equilibrium of the concentration of Phyllanthus emblica polysaccharide in the solution. The sufficient addition of rice bran protein forms an extracellular osmotic pressure higher than that inside the cell for the grafted product, promoting the precipitation of intracellular Phyllanthus emblica polysaccharide, which helps to rapidly prepare covalently grafted rice bran protein - Phyllanthus emblica polysaccharide extracellularly. Brief Description of the Drawings

[0023] 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 embodiments. Obviously, the following - described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is a statistical chart of the grafting degree of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide under different mass ratios of fresh Phyllanthus emblica fruits and rice bran protein;

[0025] Figure 2 It is a statistical chart of the grafting degree of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide under different ultrasonic power conditions;

[0026] Figure 3 It is a statistical chart of the grafting degree of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide under different extraction temperature conditions;

[0027] Figure 4 It is a statistical chart of the grafting degree of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide under different extraction time conditions;

[0028] Figure 5 It is a schematic diagram of the principle of extracellular extraction of covalently grafted rice bran protein - Phyllanthus emblica polysaccharide by one - step method of the present invention. Detailed Embodiments

[0029] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] I. Method

[0036] 1. One-step extracellular extraction of rice bran protein-phyllanthus emblica polysaccharide covalent conjugate (RBP-PEP)

[0037] Chop fresh Phyllanthus emblica and remove the shells, add an adequate amount of rice bran protein, add deionized water (the mass ratio of material to liquid is 1:35), adjust the pH to 6.5, extract for a certain period of time under the shearing at a speed of 28,000 revolutions per minute and the ultrasonic assistance in a flash extractor, filter out the waste residue, centrifuge, collect the supernatant, rotary evaporate to 1 / 4 - 1 / 5 of the original volume of the solution, add ethanol and adjust to a final concentration of 90% (volume fraction), store overnight at 4°C, centrifuge at 4000 rpm for 15 min, collect the precipitate, freeze-dry, and weigh to obtain the extraction rate. Use the physical mixture of Phyllanthus emblica polysaccharide and rice bran protein (RBP / PEP) as a control to determine the grafting degree.

[0038] Single-factor experiment: Use single-factor experiments to evaluate the effects of the ratio of Phyllanthus emblica puree to rice bran protein, ultrasonic power, extraction temperature, and extraction time on the grafting degree GD (%). The single-factor experiments are set as follows: the mass ratio of fresh Phyllanthus emblica fruit to rice bran protein (10:1, 5:1, 1:1, 1:5, 1:10); ultrasonic power (480, 560, 640, 720, 800 W); extraction temperature (50°C, 60°C, 70°C, 80°C, 90°C); extraction time (50, 100, 150, 200, 250 s). See Table 1 for details. Among them, A represents the mass ratio of fresh Phyllanthus emblica fruit to rice bran protein, B represents the ultrasonic power, C represents the extraction temperature, and D represents the extraction time.

[0039] Table 1 Extraction parameters of single-factor experiments

[0040]

[0041]

[0042] 2. Determination method of grafting degree

[0043] Determination of protein-polysaccharide grafting degree: Prepare a 0.1% (w / v) working solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS), prepare it freshly before use, and pay attention to avoiding light. Take 3 mL of 10% (w / v) sodium dodecyl sulfate (SDS) in a test tube, add 0.3 mL of the sample to the test tube. Add 1.5 mL of 0.1% (w / v) TNBS and 3 mL of 0.1 mol Na2SO3, mix the solutions evenly, incubate in a 50°C water bath in the dark for 1 h. After the reaction, let it stand in the dark at room temperature for 15 min, set the wavelength to 420 nm with a UV-visible spectrophotometer, and measure its absorbance value. Use deionized water instead of the sample as a blank control. The calculation formula for the grafting degree GD (%) is:

[0044] DG (%) = (A0 - At) / A0 × 100%;

[0045] Wherein, A0 is the absorbance value of the RBP / PEP physical mixture; At is the absorbance value of the RBP-PEP graft.

[0046] II. Results

[0047] As Figure 1 shown, with the increase in the addition amount of rice bran protein, the grafting degree of RBP-PEP gradually increases. When the ratio of Phyllanthus emblica fresh fruit to rice bran protein (A) is 1:5 (g / g), the grafting degree GD reaches the highest value (8.57%). Ultrasonic cavitation requires negative pressure to overcome the natural viscosity of the liquid in the thin region of the wave function. More solvent reduces the viscosity of the medium and the mass transfer resistance of the active substances dissolved in the solvent, thereby increasing the concentration gradient. A higher concentration gradient contributes to the site binding of Phyllanthus emblica polysaccharide and rice bran protein, which improves the grafting efficiency and thus increases the grafting degree of RBP-PEP. Subsequently, when the ratio of Phyllanthus emblica fresh fruit to rice bran protein (A) is 1:10 (g / g), the grafting degree decreases. This phenomenon can be explained as follows: (1) The increase in solvent reduces the probability of the blades of the flash extractor contacting and shearing the raw materials, thereby reducing the grafting degree of protein and polysaccharide; (2) The addition of excessive rice bran protein makes the concentration and purification processes more complex, resulting in the loss of the target product.

[0048] Figure 2 It is shown that ultrasonic power has a significant effect on the grafting degree of RBP-PEP. In the range of 480 - 640 W, the grafting degree of RBP-PEP increases significantly and reaches the highest value (8.60%) at an ultrasonic power of 640 W, and then the grafting degree decreases. This can be attributed to the physical cavitation effect and the mechanical effect generated by ultrasonic waves, which destroy the cell wall of Phyllanthus emblica, thereby enhancing the contact between rice bran protein and intracellular polysaccharide and promoting the formation of grafts through dynamic molecular penetration. However, too high ultrasonic power may lead to the degradation of the graft.

[0049] Next, the present invention observes that with the increase in the extraction temperature from 50°C to 70°C, the grafting degree of RBP-PEP increases significantly ( Figure 3 ). Increasing the extraction temperature can increase the diffusion rate and dissolution rate of polysaccharide in the liquid, and promote the Maillard reaction. However, when the extraction temperature exceeds 70°C, there is no significant difference in the PEP yield with the change of temperature (P > 0.05). Significance analysis shows that the optimal extraction temperature is 70°C. Appropriate extraction temperature can prevent energy waste and achieve a higher polysaccharide extraction rate. Moreover, the Maillard reaction by the wet heat method proceeds through the formation of covalent bonds between the amino group at the end of the protein residue and the carbonyl group of carbohydrates (monosaccharides, oligosaccharides or polysaccharides), and too high extraction temperature will produce hazards associated with thermal processing.

[0050] From Figure 4It can be observed that as the extraction time increased from 50 s to 250 s, the grafting degree of RBP-PEP increased significantly, reaching the highest value (8.57%) at an extraction time of 200 s, and then the change was no longer significant. The cavitation effects generated by the flash extractor and ultrasonic waves caused more damage to plant tissues, resulting in a higher grafting degree of RBP-PEP.

[0051] Based on the above, it can be seen that the optimal process conditions for preparing the graft of RBP-PEP by extracellular one-step ultrasonic-assisted flash extraction are as follows: the ratio of fresh Phyllanthus emblica fruits to rice bran protein is 1:5 (g / g), the ultrasonic power is 640 W, the extraction temperature is 70 °C, the extraction time is 200 s, and the corresponding grafting degree of RBP-PEP is 8.60 ± 0.31%. There is no significant difference compared with the single-factor experiment (P>0.05). The yield of the graft of RBP-PEP prepared by extracellular one-step ultrasonic-assisted flash extraction is higher than that of hot water extraction and enzyme-ultrasonic extraction. The preparation of the graft of RBP-PEP by ultrasonic-assisted flash extraction is based on the ultra-high-speed rotation of the special design head of the flash extractor and its secondary effects, as well as the cavitation effect and solubilization mechanism of ultrasonic waves ( Figure 5 ).

[0052] The flash extractor operates at maximum speed, generating significant mechanical shear force during initial extraction, instantly breaking down plant tissues into smaller particles and providing a larger mass transfer area. Meanwhile, ultrasound can also disrupt plant cell walls within seconds. Subsequently, the mass transfer barrier of Phyllanthus emblica cell walls can be more strongly disrupted. At the same time, the resonance and cavitation effects generated by ultrasound promote the penetration of the solvent into the cells, initiating the contact and dissolution of PEP. After that, due to the rotation of the blade, a negative pressure vacuum zone is formed between the inner and outer edges, resulting in ultra-dynamic molecular penetration, which accelerates the desorption and dissolution kinetics of PEP from the Phyllanthus emblica matrix under negative pressure and strong stirring. Once the PEP is released from the plant cell walls, they are quickly carried away and penetrate into the external solvent. It is worth noting that ultrasound helps this process through vibration, which not only promotes the release of polysaccharides from the cells but also helps the polysaccharides to disperse more evenly in the solvent. The strong stirring and violent vibration generated by the friction and collision between the extraction liquid driven by the flash extractor and the Phyllanthus emblica particles also contribute to the dispersion. Finally, under the high-frequency vibration of ultrasound, the rapid expansion and collapse of a large number of tiny bubbles generate temporary high temperature, high pressure, and micro-vortices in the solution, which accelerate the dissolution of polysaccharides, resulting in a rapid equilibrium of the PEP concentration in the solution. Moreover, the addition ratio of rice bran protein in the extraction system is high. In the solution system, while extracting Phyllanthus emblica polysaccharides, through specific reaction conditions, Phyllanthus emblica polysaccharides form grafts with rice bran protein extracellularly, and a concentration difference of Phyllanthus emblica polysaccharides is formed inside and outside the cells. The concentration difference of rice bran protein inside and outside the Phyllanthus emblica cells promotes the precipitation of intracellular Phyllanthus emblica polysaccharides, contributing to the rapid preparation of rice bran protein-Phyllanthus emblica polysaccharide grafts extracellularly.

[0053] Example 2

[0054] The RBP-PEP graft used in this example was prepared according to the optimal process conditions of Example 1.

[0055] 1. Determination of dough thermomechanical properties

[0056] The RBP-PEP grafts and RBP / PEP physical mixtures with different concentrations were added to the flour according to the flour ratio, and the Mixolab was used to conduct mixing experiments on the samples to explore the effects of different types of additives on the dough during stirring and baking. The results are shown in Table 2.

[0057] The thermomechanical properties of wheat blended flour with different addition amounts of RBP-PEP graft were determined using a Mixolab 2 mixer. The "Chopinp+" standard protocol mode was selected: the dough mass was set at 50 g, the target consistency (C1) was 1.1 Nm (±0.05 Nm), the kneading speed was maintained at 80 r / min, the temperature in the first stage was 30 °C and it was held for 8 min, the temperature was increased to 90 °C at a rate of 4 °C / min in the second stage and held at 90 °C for 7 min, and the temperature was decreased to 50 °C at a rate of 4 °C / min in the third stage and held at 50 °C for 5 min. Wheat flour without addition and the RBP / PEP physical mixture were selected as the control groups.

[0058] Table 2 Effects of addition of different RBP-PEP grafts and RBP / PEP mixtures on the thermomechanical properties of dough

[0059]

[0060] Note: Different superscript letters in the same column indicate significant differences (P < 0. O5).

[0061] There were significant differences (P < 0.05) in the water absorption rate of the dough between the control group and the blended flour after addition. The addition of RBP-PEP graft led to an increase in the water absorption rate of the dough, which increased with the increase in the addition amount. This can be explained that PEP is a hydrophilic polysaccharide, and after binding with RBP, it can produce a strong hydration effect, increasing the water absorption rate of the blended flour dough. The increase in the water absorption rate of the dough can not only maintain the dough moisture and reduce the cracking phenomenon caused by insufficient moisture, but also be beneficial to improving the yield rate of the flour products.

[0062] The longer the dough formation time, the longer it indicates that the dough takes to form from powder to water-absorbed and shaped. As can be seen from Table 2, the addition of RBP-PEP graft and the RBP / PEP physical mixture delayed the formation of the dough, and the graft had a greater impact on the dough. This may be because they compete with gluten proteins for water absorption during the dough mixing process, interfering with the development of the gluten protein network and resulting in a longer dough formation time.

[0063] The longer the stabilization time is, the more resistant to mechanical shearing or kneading it reflects, indicating good dough stability and strong processing resistance. As can be seen from Table 2, the addition of RBP-PEP graft and RBP / PEP physical mixture prolonged the stabilization time of the dough, and the effect of the graft was better, indicating that adding a certain amount of graft can improve the processing resistance of the dough. The C1-C2 value represents the weakening degree of gluten protein. The weakening degree reflects the strength of the gluten structure. A small weakening degree indicates strong gluten strength in the dough. It was observed from the above table that the addition of RBP-PEP graft and RBP / PEP physical mixture both showed a higher dough stabilization time and a lower C1-C2 value than the control sample, and the effect of the graft was more obvious. This indicates that the addition of RBP-PEP graft improved the dough stability and gluten strength.

[0064] Under heating conditions, C4 / C3 represents the cooking stability of the dough, and the C5-C4 value represents the degree of starch retrogradation, so as to judge the influence of additives on the starch gelatinization characteristics of the dough. As can be seen from Table 2, adding RBP-PEP graft showed a greater improvement in the cooking stability C4 / C3 of the dough than the control sample and the addition of RBP / PEP physical mixture. The C5-C4 value represents the degree of starch retrogradation, which is related to the recrystallization characteristics of starch. Compared with the control group, adding RBP-PEP graft and RBP / PEP physical mixture to the dough both reduced the C5-C4 value, and the addition of the graft had a greater impact on the C5-C4 value, stronger than directly adding the mixture, and decreased with the increase of the addition amount. The graft has the effect of inhibiting dough aging and starch retrogradation, so as to extend the shelf life of the product.

[0065] 2. Texture property analysis

[0066] The following formula was used to make sweet dough: Sweet dough formula: 1000 g of high-gluten flour, 20 g of yeast, 10 g of salt, 100 g of granulated sugar, 495 g of water, 100 g of margarine, 20 g of whole milk powder, 80 g of whole egg liquid.

[0067] RBP-PEP graft with different addition amounts was mixed into the flour according to the flour addition ratio, and sweet dough was made according to the above formula. Unadded wheat flour and RBP / PEP physical mixture were selected as the control groups. The direct method was used to make the dough. All raw materials except butter were placed in a dough mixer. First, stir at low speed for 3 min until the dough is basically formed, then stir at high speed for 4 min until the gluten network is fully formed. Then add butter and stir at low speed for 3 min to mix the butter evenly with the dough, and then stir at high speed for 4 min until the butter is fully mixed with the dough and the gluten is completely formed. Then let it stand and relax at 25 °C for 10 min. The dough was divided into 50 g portions, rounded and shaped. After the first fermentation, degassing, and the second fermentation, bake at 180 °C for 25 minutes to prepare the finished sweet bread.

[0068] Cool the sweet bread to room temperature, and use a texture analyzer to measure the texture properties of the samples, and study the effects of different additives on the index parameters of dough hardness, elasticity, adhesiveness and resilience. The results are shown in Table 3. Use a P / 100 probe for two consecutive compression tests. The pre-test speed is 1.0 mm / s, the test speed is 1.0 mm / s, the post-test speed is 3.0 mm / s, the compression ratio is 50%, the trigger force is 5 g, and the interval between two compressions is 5 s.

[0069] Table 3 Analysis of the texture properties of sweet bread with the addition of different RBP-PEP grafts and RBP / PEP mixtures

[0070]

[0071] As can be seen from Table 3, as the amount of RBP-PEP graft added gradually increases, the hardness of its sweet bread is significantly less than that without addition and with the addition of RBP / PEP mixture. The elasticity gradually increases with the addition of RBP-PEP graft, and the resilience has the same change trend as the elasticity. Among them, there are significant differences between the char siu buns treated by different freezing methods (P < 0.05), which indicates that the RBP-PEP graft has an improving effect on the organizational structure of the dough, may affect the components such as gluten protein and starch and their binding methods, and significantly improves both the elasticity and resilience, and is stronger than the mixture. Adhesiveness is a unique property of semi-solid samples, and there are differences in the adhesiveness shown among groups, which indicates that different additives will change the characteristics of sweet dough to varying degrees. In short, the RBP-PEP graft can improve the taste of sweet bread, making it softer and more elastic, and the improving effect increases with the increase of the addition amount.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracellular preparation of a covalent conjugate of rice bran protein and Phyllanthus emblica polysaccharide, characterized in that, It includes the following steps: Mix Phyllanthus emblica fresh fruits, rice bran protein and water, and under the ultrasonic assistance, use a flash extractor for extraction treatment. After that, filter to obtain a filtrate, concentrate the filtrate to obtain a concentrated solution, and then obtain a precipitate through alcohol precipitation treatment. After freeze-drying, the rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate is obtained; The ratio of the total mass of the Phyllanthus emblica fresh fruits and the rice bran protein to the mass of the water is 1:35; The mass ratio of the Phyllanthus emblica fresh fruits to the rice bran protein is 1:

5.

2. The method according to claim 1, wherein The pH of the reaction system composed of the Phyllanthus emblica fresh fruits, the rice bran protein and the water is 6.

5.

3. The method according to claim 1, wherein The concentration treatment is to concentrate the filtrate to 1 / 4 - 1 / 5 of the original volume.

4. The method according to claim 1, characterized in that, The alcohol precipitation treatment is to add ethanol to the concentrated solution to make its volume fraction 90%.

5. The method according to claim 1, characterized in that, The ultrasonic power of the ultrasonic assistance is 640 W; and / or The extraction temperature of the extraction treatment is 70 °C; and / or The extraction time of the extraction treatment is 200 s.

6. A rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate prepared by the method according to any one of claims 1-5.

7. An application of the rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate according to claim 6 in the preparation of food additives.

8. A food additive, characterized in that, It includes the rice bran protein-Phyllanthus emblica polysaccharide covalent graft conjugate according to claim 6.

9. An application of the food additive according to claim 8 in the preparation of food.

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