Preparation of a cottonseed protein-quercetin complex and its application in feed additives

Abstract

The invention discloses a preparation of a cottonseed protein-quercetin complex and its application in feed additives, including: step 1, adding cottonseed protein to deionized water, stirring under a nitrogen atmosphere, then adding hydrogen peroxide and ascorbic acid, continuing stirring to obtain a cottonseed protein mixed solution; step 2, adding quercetin to the cottonseed protein mixed solution, adjusting the pH, stirring, dialyzing to remove unreacted quercetin, freeze-drying, and obtaining a cottonseed protein-quercetin complex. The present invention uses cottonseed protein and quercetin as raw materials, adopts a free radical grafting method to prepare a cottonseed protein-quercetin complex, improves the antioxidant activity of protein, can replace antibiotics as an additive for preparing animal feed, and the raw materials of the present invention are simple and easy to obtain, the preparation method is simple, the operability is strong, and it is easy to promote.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feed additives, and more specifically, relates to the preparation of a cottonseed protein-quercetin complex and its application in feed additives. Background Art

[0002] Antibiotics, known as "feed antibiotics," are widely used as feed additives to prevent animal disease, protect animal health, promote livestock growth and production, and improve feed utilization. However, the excessive and irregular use of feed antibiotics has led to a series of issues related to livestock health and food safety, necessitating the urgent need for new feed antibiotic alternatives and solutions. Most feed antibiotic alternatives address a range of production issues and must possess antimicrobial, anti-inflammatory, antioxidant, growth-promoting, diarrhea-preventing, immune-boosting, and intestinal health-improving properties.

[0003] Cottonseed protein (CPI) is a plant-based protein extracted from cottonseed meal (CSM). As an important protein source in feed, cottonseed protein can positively impact the growth, development, immunity, and health of livestock and poultry. However, due to the interactions between various feed components and the effects of physical and biochemical factors, it is prone to self-oxidative decomposition, causing feed rancidity and reducing its nutritional value. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these objects and other advantages of the present invention, a method for preparing a cottonseed protein-quercetin complex is provided, comprising the following steps:

[0006] Step 1: adding cottonseed protein to deionized water, stirring under a nitrogen atmosphere, then adding hydrogen peroxide and ascorbic acid, and continuing to stir to obtain a cottonseed protein mixture;

[0007] Step 2: adding quercetin to the cottonseed protein mixture, adjusting the pH, stirring, dialyzing to remove unreacted quercetin, and freeze-drying to obtain a cottonseed protein-quercetin complex.

[0008] Preferably, in step 1, the mass volume ratio of cottonseed protein to deionized water is 1 g:80-200 mL.

[0009] Preferably, in step 1, the mass volume ratio of cottonseed protein, hydrogen peroxide and ascorbic acid is 1 g:1-3 mL:0.3-0.8 g.

[0010] Preferably, in the step 1, stirring is performed under a nitrogen atmosphere for 1 to 3 hours.

[0011] Preferably, in step 1, after adding hydrogen peroxide and ascorbic acid, stirring is continued for 1 to 3 hours.

[0012] Preferably, in step 2, the mass molar ratio of cottonseed protein to quercetin is 1 g:0.1-5 mmol.

[0013] Preferably, in the step 2, adjusting the pH is to adjust the pH to 8-10 using NaOH.

[0014] Preferably, in step 2, the stirring is performed at 20-30° C. for 20-30 h.

[0015] Preferably, in the step 2, freeze drying is performed at -60 to -100°C for 40 to 50 hours.

[0016] Preferably, in the step 2, before using quercetin, the quercetin is pretreated, and the specific method of the pretreatment is: quercetin, bayberry tannin and epigallocatechin gallate (EGCG) are mixed in a mass ratio of 1:0.1-0.5:0.1-0.5, stirred at 600-1200 rpm for 20-40 minutes, added to an integrated ultrasonic extrusion device for extrusion, and the extrudate is dried and ground into powder to obtain pretreated quercetin.

[0017] Preferably, in the step 2, before using quercetin, quercetin is pretreated, and the specific method of the pretreatment is: quercetin, bayberry tannin and epigallocatechin gallate are mixed in a mass ratio of 1:0.1-0.5:0.1-0.5, added to a pulsed air flow mixer, and mixed for 5-15 minutes under an intermittent jet airflow formed by nitrogen, the airflow is sprayed for 0.5-2 seconds, paused for 4-6 seconds, and the injection pressure is 0.5-2 MPa. The obtained mixture is added to an integrated ultrasonic extrusion device for extrusion, and the extrudate is dried and ground into powder to obtain pretreated quercetin.

[0018] Preferably, the screw speed of the ultrasonic extrusion integrated device is 150-250 rpm, the temperature is set as follows: zone 1 temperature 80-100°C, zone 2 temperature 90-110°C, zone 3 temperature 100-120°C, zone 4 temperature 110-130°C, head temperature 120-140°C, the residence time of the mixture in the extruder is 5-15 min, the pressure is 5-15 MPa, the ultrasonic power is 400-800 W, and the frequency is 50-60 KHz.

[0019] The invention relates to the use of the cottonseed protein-quercetin complex prepared by the preparation method as an additive in animal feed.

[0020] The present invention has at least the following beneficial effects: the present invention uses cottonseed protein and quercetin as raw materials, adopts a free radical grafting method to prepare a cottonseed protein-quercetin complex, thereby improving the antioxidant activity of the protein, providing a preliminary experimental basis for its application in feed, and can replace antibiotics as an additive for preparing animal feed. At the same time, the raw materials of the present invention are simple and easy to obtain, the preparation method is simple, the operability is strong, and it is easy to promote.

[0021] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Photos of the cottonseed protein-quercetin complexes (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention;

[0023] Figure 2 Infrared spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4), cottonseed protein (CPI), and quercetin (Q) prepared in Examples 1 to 6 of the present invention;

[0024] Figure 3 Fluorescence spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention;

[0025] Figure 4 UV spectra of the cottonseed protein-quercetin complex (Q-0.125 to Q-4), cottonseed protein (CPI), and quercetin (Q) prepared in Examples 1 to 6 of the present invention;

[0026] Figure 5 CD spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0028] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0029] Example 1

[0030] A method for preparing a cottonseed protein-quercetin complex comprises the following steps:

[0031] Step 1: Add 1 g of cottonseed protein to 100 mL of deionized water, stir for 2 h under a nitrogen atmosphere, then add 2 mL of hydrogen peroxide and 0.5 g of ascorbic acid, and stir for 2 h to obtain a cottonseed protein mixture;

[0032] Step 2: Add 0.125 mmol of quercetin to the cottonseed protein mixture, adjust the pH to 9 with NaOH, stir at 25°C for 24 hours, dialyze to remove unreacted quercetin, and freeze-dry at -80°C for 48 hours to obtain a cottonseed protein-quercetin complex, recorded as CPI-Q-0.125.

[0033] Example 2

[0034] In this example, the amount of quercetin used was 0.25 mmol, and the remaining steps were the same as in Example 1 to obtain a cottonseed protein-quercetin complex, which was recorded as CPI-Q-0.25.

[0035] Example 3

[0036] In this example, the amount of quercetin used was 0.5 mmol, and the remaining steps were the same as in Example 1 to obtain a cottonseed protein-quercetin complex, which was designated as CPI-Q-0.5.

[0037] Example 4

[0038] In this example, the amount of quercetin used was 1 mmol, and the remaining steps were the same as in Example 1 to obtain a cottonseed protein-quercetin complex, which was designated as CPI-Q-1.

[0039] Example 5

[0040] In this example, the amount of quercetin used was 2 mmol, and the remaining steps were the same as in Example 1 to obtain a cottonseed protein-quercetin complex, which was designated as CPI-Q-2.

[0041] Example 6

[0042] In this example, the amount of quercetin used was 4 mmol, and the remaining steps were the same as in Example 1 to obtain a cottonseed protein-quercetin complex, which was designated as CPI-Q-4.

[0043] The actual photos of the cottonseed protein-quercetin complex (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention are as follows: Figure 1 shown.

[0044] Figure 2 The infrared spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4), cottonseed protein (CPI) and quercetin (Q) prepared in Examples 1 to 6 of the present invention are shown in Figure 1. -1The broad peak near 2970 cm is attributed to the stretching vibration peak of -OH. -1 The peak at 1656 cm indicates the presence of CH. -1 、1540cm -1 and 1238cm -1 The peaks at 1164 cm-1 are the characteristic absorption peaks of amide I, II, and III of proteins. In the infrared spectrum of CPI-Q, the intensity of the -OH stretching vibration peak increases, which is due to the introduction of a large number of phenolic hydroxyl groups caused by the addition of quercetin. -1 and 1125cm -1 New peaks appeared, representing CO, CC, and CCO, which were attributed to the vibration of phenolic hydroxyl group and the stretching vibration of CO, respectively. The adsorption peak of amide II band shifted from 1540 cm -1 Blue shift to 1534 cm -1 This indicates that the interaction between quercetin and cottonseed protein may be related to -NH-. In summary, the cottonseed protein-quercetin complex was successfully prepared.

[0045] Figure 3 The fluorescence spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention are shown. It can be seen that CPI exhibits a strong fluorescence intensity, and the fluorescence intensity tends to decrease with increasing Q concentration. The attenuation of fluorescence intensity indicates the interaction between protein and Q, which demonstrates the obvious quenching effect of quercetin on casein tryptophan fluorescence emission in a dose-dependent manner. The maximum emission peak of the CPI-Q complex shifts from 332 nm to 334 nm, respectively. As the concentration of quercetin increases, the fluorescence intensity of the CPI-Q complex gradually decreases from 105.4 cps to 43.36 cps. This phenomenon can be attributed to the oxidation of tryptophan (Trp) residues in the protein polypeptide chain to quinones under alkaline conditions, which then undergo nucleophilic reactions with polyphenols. The reduction of Trp residues leads to a decrease in fluorescence intensity. As the concentration of quercetin increases, the phenolic hydroxyl content in the system increases, enhancing the degree of interaction between polyphenols and protein groups, forming a more stable protein-polyphenol complex with fewer exposed tryptophan residues. This blue shift phenomenon indicates that the tryptophan residues of CPI are exposed to a more hydrophilic environment, indicating the conformational unfolding of CPI.

[0046] Figure 4 UV spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4), cottonseed protein (CPI), and quercetin (Q) prepared in Examples 1 to 6 of the present invention. As can be seen, the UV absorption peak shifts to the blue with increasing Q concentration, the peak decreases, and the conjugated system becomes smaller, which may be due to the chromophore extending the protein peptide chain.

[0047] Figure 5CD spectra of cottonseed protein-quercetin complexes (Q-0.125 to Q-4) and cottonseed protein (CPI) prepared in Examples 1 to 6 of the present invention. As can be seen, the CD spectra of all samples exhibit a broad negative band around 190 to 250 nm, indicating that CPI is rich in random coil structures. As shown in Table 1, after CPI binds to Q, the α-helix and β-sheet content decreases, while the β-turn and random coil content increases, indicating that the α-helix and β-sheet content is converted to β-turns and random coils.

[0048] Table 1

[0049] CPI Q0.125 Q0.25 Q0.5 Q1 Q2 Q4 α-helix (%) 13.1 6.6 3.3 0.1 8.7 0 6.6 β-sheet (%) 48.5 28.4 30.8 33.7 20.3 33.2 26.5 β-turn(%) 4.9 15.6 13.7 14.2 16.1 14.1 14.3 Others(%) 33.4 49.5 52.2 52.0 54.9 52.7 53.2

[0050] Antioxidant activity test:

[0051] (1) DPPH free radical scavenging activity

[0052] The sample (2 mL, 1 mg / mL) was mixed with 2 mL of DPPH solution. The mixed solution was reacted in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a spectrophotometer. The DPPH radical scavenging rate was calculated using the following formula:

[0053] DPPH free radical scavenging rate (%) = (Ac-As) / Ac×100%

[0054] Where As and Ac are the absorbances of DPPH solution after adding sample solution and without adding sample solution (adding ethanol), respectively;

[0055] (2) ABTS free radical scavenging activity

[0056] 7 mM ABTS and an equal volume of 2.45 mM potassium persulfate solution were mixed to obtain an ABTS+ working solution. The solution was placed in the dark for 14 h to generate free radicals. The sample (2 mL, 1 mg / mL) was added to the ABTS+ working solution (4 mL) and shaken evenly (vortex). The mixture was reacted at room temperature in the dark for 10 min. The absorbance was measured at 734 nm using a spectrophotometer. The ABTS free radical scavenging rate was calculated using the following formula:

[0057] ABTS free radical scavenging rate (%) = (Ac-As) / Ac×100%

[0058] Where As and Ac are the absorbances of ABTS solution after adding sample solution and without adding sample solution (adding ethanol), respectively;

[0059] (3) Iron reducing ability

[0060] The sample (2 mL, 1 mg / mL) was mixed with potassium ferricyanide (1%, w / v, 1 mL), and the mixture was placed in a 50°C water bath for 20 min. A trichloroacetic acid solution (10%, w / v, 1 mL) was added. The mixture (1 mL) was mixed with deionized water (3 mL) and ferric chloride (0.1%, w / v, 0.4 mL) and reacted at room temperature for 5 min. The absorbance at 700 nm was measured using a spectrophotometer to indicate the ferric reducing ability.

[0061] (4) Hydroxyl radical scavenging activity

[0062] Take a sample (2 mL, 1 mg / mL) and mix it with 1 mL of 0.75 mmol / L o-phenanthroline solution and 2 mL of 0.2 mol / L phosphate buffer solution (pH 7.4). Then add 1 mL of 0.75 mmol / L ferrous sulfate solution and 1 mL of 0.01% hydrogen peroxide solution. After mixing, place it in a 37°C water bath for 1 hour and measure the absorbance at 536 nm. The control group uses distilled water instead of hydrogen peroxide solution, and the blank group uses distilled water instead of sample. The calculation formula is as follows:

[0063] Hydroxyl radical scavenging rate (%) = (As-Ac) / (Ab-Ac) × 100%

[0064] Where As is the absorbance of the experimental sample group; Ac is the absorbance of the blank group; Ab is the absorbance of the control group.

[0065] The antioxidant activities of the cottonseed protein-quercetin complexes and cottonseed protein prepared in Examples 1 to 6 are shown in Table 2. It can be seen that the DPPH free radical scavenging activity, ABTS free radical scavenging activity, iron reducing ability, and hydroxyl free radical scavenging activity of the cottonseed protein-quercetin complexes prepared in Examples 1 to 6 of the present invention are all higher than those of cottonseed protein, indicating that the present invention successfully improves the antioxidant activity of protein. This is attributed to the fact that quercetin contains many hydroxyl groups, especially phenolic hydroxyl groups, which have strong antioxidant capacity.

[0066] Table 2

[0067]

[0068] Example 7

[0069] In this embodiment, before using quercetin, quercetin was pretreated, and the specific method of the pretreatment was as follows: quercetin, bayberry tannin and epigallocatechin gallate were mixed in a mass ratio of 1:0.3:0.2, stirred at 800 rpm for 30 min, added to an integrated ultrasonic extrusion device for extrusion, and the extrudate was dried and ground into powder to obtain pretreated quercetin; wherein the screw speed of the integrated ultrasonic extrusion device was 200 rpm, the temperature was set to: 90° C. in zone 1, 100° C. in zone 2, 110° C. in zone 3, 120° C. in zone 4, and a head temperature of 130° C.; the mixture stayed in the extruder for 10 min, the pressure was 10 MPa, the ultrasonic power was 600 W, and the frequency was 55 kHz; the remaining steps were the same as in Example 6.

[0070] Example 8

[0071] In this embodiment, before using quercetin, quercetin was pretreated, and the specific method of the pretreatment was as follows: quercetin, bayberry tannin and epigallocatechin gallate were mixed in a mass ratio of 1:0.3:0.2, added to a pulsed air flow mixer, and mixed for 10 minutes under an intermittent jet airflow formed by nitrogen, wherein the airflow was sprayed for 1 second and paused for 5 seconds, and the injection pressure was 1 MPa. The obtained mixture was added to an ultrasonic extrusion integrated device for extrusion, and the extrudate was dried and ground into powder to obtain pretreated quercetin; wherein the screw speed of the ultrasonic extrusion integrated device was 200 rpm, the temperature was set to: 90° C. in zone 1, 100° C. in zone 2, 110° C. in zone 3, 120° C. in zone 4, and a head temperature of 130° C. The mixture stayed in the extruder for 10 minutes, the pressure was 10 MPa, the ultrasonic power was 600 W, and the frequency was 55 kHz; and the remaining steps were the same as in Example 6.

[0072] In Examples 7 and 8, bayberry tannin and epigallocatechin gallate were added to quercetin, and the mixture was extruded using an integrated ultrasonic extrusion device. In Example 8, a pulsed airflow mixer was used to make the materials more evenly mixed, both of which helped to increase the total phenol content and the number of hydroxyl groups in the obtained composite, thereby further enhancing its antioxidant activity. The antioxidant activity test results are shown in Table 3. Compared with Example 6, Examples 7 to 8 were all improved.

[0073] Table 3

[0074] Example 6 Example 7 Example 8 DPPH free radical scavenging rate (%) 80.3 85.6 87.2 ABTS free radical scavenging rate (%) 98.1 99.0 99.4 Iron reducing capacity 0.38 0.41 0.43 Hydroxyl radical scavenging rate (%) 70.2 74.5 76.7

[0075] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A cottonseed protein-quercetin complex as an additive in animal feed, characterized in that: The preparation method of the cottonseed protein-quercetin complex comprises the following steps: Step 1: Add cottonseed protein to deionized water, stir for 1-3 hours under a nitrogen atmosphere, then add hydrogen peroxide and ascorbic acid, and continue stirring for 1-3 hours to obtain a cottonseed protein mixture; wherein the mass volume ratio of cottonseed protein to deionized water is 1 g:80-200 mL; and the mass volume ratio of cottonseed protein, hydrogen peroxide, and ascorbic acid is 1 g:1-3 mL:0.3-0.8 g; Step 2: adding quercetin to the cottonseed protein mixture, adjusting the pH, stirring at 20-30° C. for 20-30 h, dialyzing to remove unreacted quercetin, and freeze-drying to obtain a cottonseed protein-quercetin complex; wherein the pH is adjusted to 8-10 with NaOH; freeze-drying is drying at -60--100° C. for 40-50 h; and the mass molar ratio of cottonseed protein to quercetin is 1 g:0.1-5 mmol; The step 2 also includes pre-treating the quercetin before using it. The specific method of the pre-treatment is: mixing quercetin, bayberry tannin and epigallocatechin gallate in a mass ratio of 1:0.1-0.5:0.1-0.5, adding them to a pulse air flow mixer, mixing them for 5-15 minutes under an interval jet air flow formed by nitrogen, jetting the air flow for 0.5-2 seconds, pausing for 4-6 seconds, and the jet pressure is 0.5-2 MPa, adding the obtained mixture to an ultrasonic extrusion integrated device for extrusion, and The extrudate is dried and ground into powder to obtain pretreated quercetin; wherein, the screw speed of the integrated ultrasonic extrusion device is 150-250 rpm, the temperature is set as follows: zone 1 temperature 80-100°C, zone 2 temperature 90-110°C, zone 3 temperature 100-120°C, zone 4 temperature 110-130°C, and the head temperature is 120-140°C. The residence time of the mixture in the extruder is 5-15 minutes, the pressure is 5-15 MPa, the ultrasonic power is 400-800 W, and the frequency is 50-60 kHz.

2. A use of the cottonseed protein-quercetin complex as claimed in claim 1 as an additive in animal feed, characterized in that: The cottonseed protein-quercetin complex has a DPPH free radical scavenging rate greater than 55%, an ABTS free radical scavenging rate greater than 78%, an iron reducing capacity greater than 0.18, and a hydroxyl free radical scavenging activity greater than 50%.

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