Wheat bran phenolic acid-protein non-covalent compound and preparation method thereof

Abstract

The invention discloses a wheat bran phenolic acid-protein non-covalent compound and a preparation method thereof. According to the method disclosed by the invention, covalent linkage of bound phenolic acid and arabinoxylan in wheat bran is broken through an alkali-solution and acid-precipitation method, phenolic acid and protein are synchronously released, and non-covalent compounding of phenolic acid and protein is realized through a green and safe pH driving method. The compound is synchronously prepared in the synchronous extraction process of phenolic acid and protein, so that the activity of natural phenolic acid in wheat bran can be better maintained; the preparation method is simple and easy to implement, the purposes of synchronously releasing phenolic acid and protein and extracting the wheat bran phenolic acid-protein non-covalent compound in one step are achieved, the cost can be reduced, the time can be shortened, and remarkable economic and time advantages are achieved. The total phenolic acid extraction rate of the wheat bran bound phenol-protein compound prepared by the method reaches 28.8%, and the wheat bran bound phenol-protein compound has high application value and is especially suitable for functional material development in the fields of food, medicine and the like.

Description

Technical Field

[0001] The invention relates to a wheat bran phenolic acid-protein non-covalent complex and a preparation method thereof, and belongs to the technical field of food processing. Background Art

[0002] Wheat bran is a byproduct of wheat processing. As a natural plant resource, it is rich in nutrients such as dietary fiber, protein, fat, etc., and contains a large number of phenolic substances. These ingredients not only have important nutritional value, but are also widely used in food, health products and medicine. Studies have found that phenolic acids in wheat bran mainly exist in a bound state, accounting for 80-90% of the total phenolic acid content, which gives it huge development potential. However, most of them are connected to arabinoxylan by ester bonds, and their structure is stable and not easy to be released, resulting in low bioavailability, which limits its application in functional food and medicine. Therefore, it is necessary to use certain processing methods to achieve the purpose of effectively releasing bound phenolic acids in wheat bran.

[0003] At present, the commonly used methods for extracting phenolic acids from wheat bran mainly include solvent extraction, enzymatic hydrolysis and alkaline hydrolysis. However, these methods often require the use of organic solvents, chemical reagents or high temperature and high pressure conditions, which will not only affect the stability of phenolic acids, but also may introduce exogenous pollutants, reducing their safety and biological activity. The protection and stability improvement of natural phenolic acids also face great challenges. Phenolic acids and proteins form complexes through non-covalent bonds, which can not only improve their stability, but also enhance their antioxidant activity. At the same time, proteins can also protect phenolic acids from oxidative degradation.

[0004] However, currently, when preparing phenolic acid-protein complexes, phenolic acid and protein are prepared separately, and after each is dissolved, the phenolic acid solution is added to the protein solution to prepare the phenolic acid-protein complex. This method cannot overcome the problem of decreased stability of phenolic acid caused by the preparation method, and cannot ensure that phenolic acid and protein are effectively combined together. For example, in the preparation method of a pecan cattail phenolic acid-pea protein complex with starch digestive enzyme inhibitory activity disclosed in patent CN118303622A, pecan cattail phenolic acid and pea protein are prepared separately, and then the complex is prepared. This method has a complex process, a low yield of the complex, and the preparation process has a certain impact on the stability of phenolic acid and the stability of protein. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention prepares a wheat bran phenolic acid-protein non-covalent complex and a preparation method thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing a wheat bran phenolic acid-protein non-covalent complex, comprising the following steps:

[0008] S1, adding an alkaline solution with a concentration of 0.10-0.15M to wheat bran, stirring evenly and heating to react, and separating to obtain a supernatant after the reaction;

[0009] S2, adjusting the pH of the supernatant in step S1 to 3-4, allowing it to settle, and separating to obtain a precipitate;

[0010] S3. Redissolving the precipitate obtained in step S2 and adjusting the pH to neutral to obtain the wheat bran phenolic acid-protein non-covalent complex.

[0011] In one embodiment of the present invention, in step S1, the solid-liquid ratio of wheat bran to alkaline solution is 1:10 to 1:20 in grams per milliliter.

[0012] In one embodiment of the present invention, the alkaline solution is a NaOH solution.

[0013] In one embodiment of the present invention, in step S1, the reaction is carried out at 45-55° C. for 1-3 hours.

[0014] In one embodiment of the present invention, in step S1, separation is performed by centrifugation at 10,000 to 14,000 r / min for 5 to 15 minutes.

[0015] In one embodiment of the present invention, in step S2, the static precipitation is performed at 20 to 30° C. for 20 to 60 minutes.

[0016] In one embodiment of the present invention, in step S2, separation is performed by centrifugation at 8000-12000 r / min for 5-15 minutes.

[0017] In one embodiment of the present invention, in step S3, the pH is adjusted to neutral and then freeze-dried to obtain the wheat bran phenolic acid-protein non-covalent complex.

[0018] The second object of the present invention is to provide a wheat bran phenolic acid-protein non-covalent complex prepared by the preparation method.

[0019] The third object of the present invention is to provide the use of the wheat bran phenolic acid-protein non-covalent complex in food, medicine or health care products.

[0020] Beneficial effects of the present invention:

[0021] The present invention breaks the covalent connection between bound phenolic acid and arabinoxylan in wheat bran by high-concentration alkali dissolution and acid precipitation, releases them synchronously with proteins, and realizes non-covalent compounding of the two by a green and safe pH-driven method. The wheat bran phenolic acid-protein non-covalent complex prepared by the present invention is a process of synchronously preparing the complex during the synchronous extraction of phenolic acid and protein, which can better maintain the activity of natural phenolic acid in wheat bran, enhance the stability of phenolic acid and improve its bioavailability through the protective effect of protein on phenolic acid; at the same time, avoid exogenous addition of protein and exogenous impurity pollution; the preparation method of the present invention is simple and easy to implement, achieves the purpose of synchronously releasing phenolic acid and protein, and extracting the wheat bran phenolic acid-protein non-covalent complex in one step, which can reduce costs and shorten time, and has significant economic and time advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The effect of different alkaline dissolution pH on the quality of wheat bran phenolic acid-protein non-covalent complex;

[0024] Figure 2 The effect of different alkaline dissolution pH on the protein purity of wheat bran phenolic acid-protein non-covalent complex;

[0025] Figure 3 The effect of different alkaline solution pH on the total protein extraction rate;

[0026] Figure 4 The effect of different alkaline solution pH on the extraction rate of total phenolic acids;

[0027] Figure 5 The effect of different alkali concentrations on the quality of wheat bran phenolic acid-protein non-covalent complexes;

[0028] Figure 6 The effect of different alkali concentrations on the purity of protein in the wheat bran phenolic acid-protein non-covalent complex;

[0029] Figure 7 The effect of different alkali solution concentrations on the total protein extraction rate;

[0030] Figure 8 The effect of different alkali solution concentrations on the extraction rate of total phenolic acids;

[0031] Fig. 9The effect of different acid precipitation pH on the quality of wheat bran phenolic acid-protein non-covalent complex;

[0032] Fig.10 The effect of different acid precipitation pH on the protein purity of wheat bran phenolic acid-protein non-covalent complex;

[0033] Fig.11 The effect of different acid precipitation pH on the total protein extraction rate;

[0034] Fig.12 The effect of different acid precipitation pH on the extraction rate of total phenolic acids;

[0035] Fig.13 This is the infrared spectrum of wheat bran phenolic acid-protein non-covalent complex and wheat bran protein;

[0036] Fig.14 The peak fitting curves of wheat bran protein (a) and wheat bran phenolic acid-protein non-covalent complex (b) amide I band;

[0037] Fig.15 is the secondary structure content of wheat bran protein and wheat bran phenolic acid-protein non-covalent complex;

[0038] Fig.16 The effect of different blockers on the fluorescence intensity of wheat bran phenolic acid-protein non-covalent complex. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the claims attached to the application.

[0040] Source of raw materials

[0041] Wheat bran was sourced from Yushi Workshop, Shangqiu (Henan, China). Caffeic acid, p-coumaric acid, and ferulic acid standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. In HPLC determination, methanol, acetonitrile, and acetic acid used as mobile phases were all chromatographic grade reagents purchased from Merck KGaA. The water used as mobile phase was obtained from an ultrapure water system (Merck MILLIPORE MILLI-Q Reference). In addition, deionized water was used throughout the experiment, and all reagents used were of analytical grade.

[0042] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased from commercial sources, or prepared by conventional methods, or are commonly used in the industry.

[0043] Embodiment 1:

[0044] The determination of phenolic acid content in wheat bran raw material comprises the following steps:

[0045] Weigh 1g of wheat bran raw material, add 50mL of pre-cooled 80% acetone, mix well, shake on a shaker at room temperature for 10min, then centrifuge at 4℃ and 8000rpm for 10min, discard the supernatant, mix the wheat bran residue with 20mL NaOH (4M), digest at room temperature for 1h, acidify to pH=2 with 2M HCl, then add 50mL of ether, invert several times to mix thoroughly, centrifuge at room temperature at 1000rpm for 10min, separate the organic phase and the aqueous phase. Collect the supernatant, and add 25mL of ethyl acetate to further extract the residue. Mix the supernatants extracted with ether and ethyl acetate, evaporate to dryness at 45℃, add pre-cooled methanol to redissolve, make the volume to 10mL, and determine the phenolic acid content by high performance liquid chromatography.

[0046] This example determines the types and contents of bound phenols in wheat bran raw materials.

[0047] Table 1 Regression equations, correlation coefficients and contents of three phenolic acids in wheat bran raw materials

[0048] Phenolic acid types Standard curve <![CDATA[Correlation coefficient (R 2 )]]> Content in wheat bran raw material (mg / g) Caffeic acid y=28.69x-68.65 0.9982 0.059 p-Coumaric acid y=38.09x-38.76 0.9991 0.077 Ferulic acid y=29.89x-50.18 0.9990 1.803

[0049] Embodiment 2:

[0050] The conventional alkali dissolution and acid precipitation method was used to prepare the wheat bran phenolic acid-protein non-covalent complex:

[0051] The alkali dissolution and acid precipitation method was used to prepare the solution with a material-water ratio (g / mL) of 1:15. 20g of wheat bran raw material was weighed in a beaker, 300mL of deionized water was added, and a rotor was placed for magnetic stirring to make the solution uniform. 1M NaOH solution was gradually added dropwise to adjust the pH to 8, 9, 10, 11, and 12, respectively. Then, it was placed in a 50°C constant temperature water bath for 2 hours, centrifuged at 12000rpm at room temperature for 10min, the supernatant was retained, and the pH was adjusted to 4.6 with 1M HCl, then allowed to stand for 30min, centrifuged at 10000rpm at room temperature for 10min, the precipitate was taken and re-dissolved with a small amount of water, the pH was adjusted to 7, and the sample obtained after freeze-drying was stored in a -20°C refrigerator for use.

[0052] This example studies the effect of different alkaline solution pH on the purity of wheat bran phenolic acid-protein complex and the total protein extraction rate. Figure 1 and Figure 3It can be seen that as the pH increases, the mass of the obtained complex increases, reaching about 1.45g at pH = 12; the total protein extraction rate also continues to increase, eventually reaching about 80%. Alkaline conditions help break the binding of protein with other substances in wheat bran, making the protein easier to release and dissolve. The effect of different alkaline dissolution pH on the purity of protein in the complex is shown in Figure 2. Figure 2 As shown in the figure, the broken line shows that as the pH increases from 8 to 12, the protein purity in the sample increases and tends to be stable. This may be because during the extraction process, impurities such as weak acidic substances may precipitate or bind to solid residues, thereby being effectively removed. As the pH increases, these impurities are more likely to condense into particles or precipitate, which helps to obtain a purer sample. However, Figure 4 It can be found that the phenolic acid extraction rate of the sample prepared at this time is extremely low. It can be seen that the phenolic acid-protein non-covalent complex prepared by the conventional alkali dissolution and acid precipitation method has extremely low phenolic acid content and high protein content, and cannot fully utilize the phenolic acid active substances in wheat bran. It is speculated that the effect of effectively releasing phenolic acid cannot be achieved by adjusting the pH alone during the alkali dissolution process, and further exploration is needed to improve the phenolic acid extraction rate and the yield of the complex.

[0053] Embodiment 3:

[0054] The method for preparing the wheat bran phenolic acid-protein non-covalent complex comprises the following steps:

[0055] The improved alkali dissolution and acid precipitation method was used to prepare the solution with a material-water ratio (g / mL) of 1:15. 20g of wheat bran raw material was weighed in a beaker, and 300mL of alkali solution of different concentrations was added respectively. The solution was stirred magnetically to make it uniform. Then it was placed in a 50℃ constant temperature water bath for 2 hours, centrifuged at 12000rpm for 10min at room temperature, and the supernatant was retained. The solution was adjusted to different pH values ​​with 1M HCl, and then allowed to stand for 30min, centrifuged at 10000rpm for 10min at room temperature, and the precipitate was taken and re-dissolved with a small amount of water, and the pH was adjusted to 7. After freeze-drying, the obtained wheat bran phenolic acid-protein non-covalent complex was stored in a -20℃ refrigerator for use.

[0056] When the sample was prepared by the above-mentioned alkali dissolution and acid precipitation method, the total phenolic acid extraction rate and the total protein extraction rate were used as indicators, and the alkali solution concentration (0.1M, 0.125M, 0.15M, 0.175M, 0.2M) and the acid precipitation pH (5.8, 5.2, 4.6, 4.0, 3.4, 2.2) during the extraction process were analyzed by single factor experiment. When a single factor was explored, the levels of the other factors were set as alkali solution concentration 0.15M NaOH and acid precipitation pH = 4.6.

[0057] The protein purity of the obtained sample was determined using a Heineng K1110 Kjeldahl nitrogen analyzer. A standard sulfuric acid titration solution [c(1 / 2H2 SO 4 =0.1000mol / L)], 40%, 15% and 5% NaOH solution, 2% boric acid solution, and then weigh 0.1g methyl red and 0.1g bromocresol green respectively, and dilute to 100mL with anhydrous ethanol. Mix them at a ratio of 1:5 before use, and then mix them with boric acid solution at a ratio of 1:100 to obtain a methyl red and bromocresol green mixed indicator.

[0058] Take 0.2g of sample and add it to the digestion tube, then add 10mL of concentrated sulfuric acid, 0.2g of anhydrous copper sulfate and 3g of anhydrous potassium sulfate, then put the digestion tube into the digestion furnace, connect the waste discharge device, and start the digestion process. The experiment must be carried out in a fume hood throughout the experiment. After the digestion is completed, the sample is clear and transparent blue-green and can be measured. The total protein extraction rate is calculated according to the following formula:

[0059]

[0060] x 0 : Mass of wheat bran phenolic acid-protein complex extracted from 1g wheat bran

[0061] p: Purity of the obtained complex (%)

[0062] X: Protein content in 1g of wheat bran raw material

[0063] This example studies the effects of different alkali concentrations and acid precipitation pH on the prepared wheat bran phenolic acid-protein non-covalent complex. Figure 5 and Figure 6 It can be seen that with the increase of alkali solution concentration, the mass of the obtained wheat bran phenolic acid-protein non-covalent complex gradually increases. However, starting from 0.15M NaOH, the protein purity decreases significantly. Under 0.2M NaOH conditions, the protein purity of the complex obtained is only about 20%. This may be because under excessively high alkaline conditions (such as 0.2M NaOH), the dissociation and partial precipitation of proteins and other components will be induced. At this time, due to the interaction and aggregation of substances such as proteins, xylan and phenolic acids, larger particles may be formed, which promotes the occurrence of flocculation, making the extracted protein product present a granular or uneven morphology, affecting its purity.

[0064] Depend on Fig. 9 and Fig.10 It can be seen that as the acid precipitation pH decreases, the mass of the obtained complex continues to increase, and its purity is highest when the acid precipitation pH = 4.6. When the pH changes to both sides, the purity decreases. This is because pH = 4.6 just corresponds to the isoelectric point of wheat gluten protein. In addition, it is found that when the pH changes to acidic conditions, the decrease in protein purity is smaller than that under alkaline conditions, and can be maintained at 60% to 70%.

[0065] Embodiment 4:

[0066] A method for preparing a wheat bran phenolic acid-protein non-covalent complex comprises the following steps:

[0067] The samples obtained by freeze-drying in Examples 2 and 3 were enzymatically hydrolyzed to remove proteins therein. A certain amount of sample was weighed, 0.4% alkaline protease was added, 50 mL of ultrapure water was added and the pH was adjusted to 8.5. Then, the sample was placed in a 60°C constant temperature water bath for enzymatic hydrolysis for 90 minutes. After the enzymatic hydrolysis was completed, the sample was centrifuged at 10000 rpm at room temperature for 10 minutes, and the supernatant was retained and stored in a 4°C refrigerator for later use.

[0068] HPLC was used to determine the phenolic acid content in wheat bran raw materials and wheat bran phenolic acid-protein non-covalent complexes. First, the phenolic substance extract was filtered through a 0.22μm filter membrane for testing. Caffeic acid, p-coumaric acid and ferulic acid were selected as standard substances. Weigh 1 mg of each, dissolve it with chromatographic grade methanol and dilute it to a 10mL brown volumetric flask, then dilute each standard solution with chromatographic grade methanol to 100, 50, 25, 12.5, 6.25, and 3.125μg / mL and filter it through a 0.22μm filter membrane for use. The experiment used an Agilent 1260HPLC system, and the chromatographic column used a Zorbax SB-C18 analytical column (250×4.6mm, id., 5μm, USA). The mobile phases were: solvent A (0.4% acetic acid, v / v); solvent B (acetonitrile), the flow rate was set to 1.0mL / min, the column temperature was 25℃, the measurement wavelength was 280nm, and the injection volume was 10μL. The gradient elution program is as follows: from 0 to 40 min, the proportion of solvent B in the mobile phase increases from 5% to 25%, from 40 to 45 min, the proportion of solvent B in the mobile phase increases from 25% to 35%, from 45 to 50 min, the proportion of solvent B in the mobile phase increases from 35% to 50%, and from 50 to 55 min, the proportion of solvent B in the mobile phase decreases from 50% to 5%.

[0069]

[0070] x 咖 +x 对 +x 阿 : The sum of caffeic acid, p-coumaric acid and ferulic acid in the complex extracted from 1g of wheat bran raw material

[0071] X: The sum of caffeic acid, p-coumaric acid and ferulic acid in 1g of wheat bran raw material

[0072] This example studies the changes in the extraction rates of total protein and total phenolic acid under different extraction conditions. Figure 7 and 8The changes in the extraction rates of total protein and total phenolic acid under different alkali concentrations were shown. The total protein extraction rate remained at a high level under 0.1, 0.125, and 0.15 M NaOH conditions, and was significantly different from the extraction rates under other concentrations; while total phenolic acid showed a higher extraction rate under 0.15 M NaOH and higher alkali concentrations. In summary, 0.1, 0.125, and 0.15 M NaOH concentrations may be one of the ideal conditions for effective extraction of total protein because they can achieve a higher total protein extraction rate. However, considering the extraction of total phenolic acid, the 0.15 M NaOH condition was finally selected to further improve the extraction rate.

[0073] Fig.11 and Fig.12 The effect of different acid precipitation pH on the extraction rate of total protein and total phenolic acid is shown. It can be seen from the figure that from pH = 4.0 to 2.2, the extraction rates of total phenolic acid and total protein are both high, and there are significant differences compared with other acid precipitation pH conditions. However, if the optimal acid precipitation pH conditions are to be determined, it is necessary to consider the purity of the obtained protein comprehensively. pH = 4.0 and 3.4 are better choices. For total protein and total phenolic acid, when pH = 3.4, the extraction rates of both are higher than when pH = 4.0. Therefore, the optimal conditions for subsequent extraction of wheat bran phenolic acid-protein non-covalent complexes are finally determined to be: alkali concentration 0.15M NaOH, acid precipitation pH = 3.4. So far, the wheat bran phenolic acid-protein non-covalent complex prepared by this method has a higher yield than the traditional alkali dissolution and acid precipitation method, and successfully releases more phenolic acids in wheat bran and combines with its own wheat bran protein, while maintaining the protein purity of the complex at a good level.

[0074] Embodiment 5:

[0075] Weigh 2 mg of freeze-dried powder of wheat bran phenolic acid-protein non-covalent complex, mix with potassium bromide at a ratio of 1:100, grind thoroughly in a mortar, and press into tablets. Scan using a Fourier transform infrared spectrometer to obtain the infrared spectrum of the sample. Detection conditions: room temperature, dry environment. Scanning range: 4 000~400cm -1 , resolution 4cm -1 , accumulated 32 times. The infrared spectrum data were analyzed using Peakfit software. First, baseline correction and Fourier self-deconvolution were performed, and then the spectrum was smoothed, denoised and second-order derivative was processed. The secondary structure of the obtained sub-peak position was identified, and the spectrum was fitted with Gaussian curves multiple times according to the peak position of each sub-peak to minimize the residual error, and then the percentage of each conformational component was calculated from the area of ​​each sub-peak.

[0076] The changes in the secondary structure of proteins are mainly reflected in the amide I band and the amide II band. The infrared spectrum is shown in Fig.13 As shown in the figure, wheat gluten protein is at 1656cm-1 and 1541cm -1 The vibration bands of amide I and amide II are shown at the bottom and bottom of the graphene. After forming a complex with phenolic acid, the peak positions shifted to a certain extent, and the peak intensity decreased compared with that of gluten protein. This indicates that hydrogen bonding and hydrophobic interaction may be involved in the binding of the two, and structural changes have occurred in the complex. Such changes may include adjustments in protein conformation, changes in vibration frequency caused by new interactions between groups, etc. The absorption peak of the complex is between 3200 and 3600 cm -1 The characteristic band is the amide A band, which represents the hydrogen bond vibration between the amino and carboxyl groups in the protein. It can be seen that the gluten protein and phenolic acid interact to form hydrogen bonds.

[0077] Located between 1600 and 1700 cm -1 The content of each sub-peak in the amide I band can reflect the changes in the secondary structure of the protein. Among them, the β-fold is located at 1600-1640 cm -1 , irregular curl at 1640~1650cm -1 , α-helix is ​​located at 1650~1660cm -1 , β-turn angle is located at 1660~1700cm -1 The amide I band in the infrared spectrum was baseline corrected, smoothed and Fourier self-deconvolved using Omnic 8.0 and Peakfit 4.12 software, and the spectrum was fitted after the second-order derivative of the self-deconvolved spectrum, as shown in Figure 2. Fig.14 , obtain each sub-peak of the amide I band and attribute the peaks, calculate the peak area percentage of each sub-peak, and determine the percentage of the secondary structure content, such as Fig.15 shown.

[0078] After fitting the amide I band peaks of gluten protein and wheat bran phenolic acid-protein non-covalent complex, the contents of each secondary structure were obtained. Compared with gluten protein, the β-sheet and β-turn contents of the complex decreased, while the α-helix and random coil contents increased. This indicates that in the process of binding between gluten protein and phenolic acid, some original hydrogen bonds may be destroyed, affecting the original β-sheet structure of the protein, thereby reducing its content. At the same time, new hydrogen bonds may promote the appearance of α-helix and random coil structures and increase their contents. The increase in α-helix content may also be caused by the binding of protein and phenolic acid through electrostatic and hydrophobic interactions, which can enhance the stability of the protein.

[0079] Embodiment 6:

[0080] 0.5 mg / mL aqueous solutions of wheat gluten protein and wheat bran phenolic acid-protein non-covalent complex were prepared, and the fluorescence intensity of wheat gluten protein and wheat bran phenolic acid-protein non-covalent complex was measured at an excitation wavelength of 280 nm, an emission wavelength of 300-500 nm, and a slit width of 5 nm. Chemical bond blockers such as deionized water, SDS, Urea, and NaCl solution (final concentration of 10 mmol / L) were added to the system for reaction, and the emission fluorescence spectrum was scanned at an excitation wavelength of 280 nm.

[0081] observe Fig.16 It can be seen that compared with the control group, the SDS group has the largest fluorescence intensity, followed by the Urea group, and the NaCl group has the lowest fluorescence intensity. This is because the addition of SDS destroys the non-covalent interaction between gluten protein and phenolic acid, especially the hydrophobic interaction. Since the structure of protein in deionized water is relatively compact, and SDS makes it more unfolded, the interaction between phenolic acid and protein is reduced, resulting in an enhancement of the fluorescence signal. NaCl mainly affects the electrostatic interaction and has less damage to the protein structure, so the change in fluorescence intensity is relatively mild. The denaturing effect of urea will cause the partial unfolding of the protein structure, exposing the aromatic amino acids and increasing the fluorescence intensity, but it is usually not as significant as SDS.

[0082] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a wheat bran phenolic acid-protein non-covalent complex, characterized in that: The steps include: S1, adding an alkaline solution with a concentration of 0.10-0.15M to wheat bran, stirring evenly and heating to react, and separating to obtain a supernatant after the reaction; S2, adjusting the pH of the supernatant in step S1 to 3-4, allowing it to settle, and separating to obtain a precipitate; S3. Redissolving the precipitate obtained in step S2 and adjusting the pH to neutral to obtain the wheat bran phenolic acid-protein non-covalent complex.

2. The preparation method according to claim 1, characterized in that: In step S1, the solid-liquid ratio of wheat bran to alkaline solution is 1:10 to 1:20 in terms of grams per milliliter.

3. The preparation method according to claim 2, characterized in that: The alkaline solution is a NaOH solution.

4. The preparation method according to claim 1, characterized in that: In step S1, the reaction is carried out at 45 to 55° C. for 1 to 3 hours.

5. The preparation method according to claim 1, characterized in that: In step S1, separation is performed by centrifugation at 10,000 to 14,000 r / min for 5 to 15 minutes.

6. The preparation method according to claim 1, characterized in that: In step S2, the static precipitation is performed at 20 to 30° C. for 20 to 60 minutes.

7. The preparation method according to claim 1, characterized in that: In step S2, separation is performed by centrifugation at 8000-12000 r / min for 5-15 minutes.

8. The preparation method according to claim 1, characterized in that: In step S3, the pH is adjusted to neutral and then freeze-dried to obtain the wheat bran phenolic acid-protein non-covalent complex.

9. A wheat bran phenolic acid-protein non-covalent complex prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the wheat bran phenolic acid-protein non-covalent complex according to claim 9 in food, medicine or health care products.

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