Preparation method of rice peptide for enhancing immunity

Abstract

The application relates to the technical field of rice peptide, and discloses a preparation method of rice peptide with enhanced immunity, which comprises the following steps: sorting and crushing; defatting: defatting of rice residue powder is carried out by adopting an organic solvent leaching method, steam desolventizing is adopted after defatting is completed, and defatted rice residue powder is obtained after solid cleaning and drying; soluble sugar separation: defatted rice residue powder is added into deionized water to configure into a slurry, alpha-amylase is added, starch is enzymolyzed at 60-70 DEG C and pH 6.0-6.5, and the enzyme is inactivated after enzymolysis is completed; the slurry concentration is adjusted to 10%, supernatant is obtained by centrifugation, soluble sugar separation is carried out by using a nanofiltration membrane, and a cut-off liquid is prepared; enzymolysis; membrane filtration purification; and drying: vacuum freeze drying is carried out to obtain rice peptide. The preparation method of the rice peptide with enhanced immunity is adopted, the content of reducing sugar is reduced, browning is inhibited, the quality of the rice peptide is improved, and the prepared rice peptide has the effect of enhancing immunity.

Description

Technical Field

[0001] This invention relates to the field of rice peptide technology, and in particular to a method for preparing rice peptides that enhance immunity. Background Technology

[0002] Peptides are a high-quality protein source suitable for various populations to supplement their protein intake. They not only possess advantages such as easy digestibility and absorption, low allergenicity, low osmotic pressure, and high solubility, but also have various physiological regulatory functions. Rice protein peptides are generally extracted from byproducts of rice processing, primarily rice residue and rice bran. Rice residue, a byproduct of rice starch (syrup) processing, is rich in protein (40%-70%), with a higher protein content than rice bran (12%-17%). Rice residue and rice bran, rich in rice protein, are mostly used as animal feed, resulting in low industrial added value and significant resource waste. With ongoing research into rice protein peptides, they have been discovered to be a plant protein resource with extremely high nutritional value.

[0003] The main methods for preparing rice protein peptides are enzymatic hydrolysis and microbial fermentation. Enzymatic hydrolysis is more suitable for the industrial production of rice protein peptides and is currently the most commonly used method in both production and research. However, browning may occur during enzymatic hydrolysis, which can affect the quality of the rice peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing rice peptides that enhance immunity, thereby solving the aforementioned technical problems. This invention improves the quality of rice peptides by reducing the content of reducing sugars and inhibiting browning, while simultaneously producing rice peptides that enhance immunity.

[0005] To achieve the above objectives, the present invention provides a method for preparing rice peptides that enhance immunity, comprising the following steps:

[0006] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it with distilled water 2-3 times, dry it for later use, crush the dried rice residue and pass it through a sieve of 60-80 mesh to obtain rice residue powder.

[0007] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0008] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry, add α-amylase, and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme; adjust the slurry concentration to 10%, centrifuge and collect the supernatant, use a nanofiltration membrane to separate soluble sugars, and collect the retentate for later use;

[0009] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6-8%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15-20 minutes, and collect the supernatant for later use.

[0010] Step 5, membrane filtration purification: membrane filtration is performed sequentially using ultrafiltration membrane and nanofiltration membrane, and finally the retentate is collected;

[0011] Step 6, Drying: The retentate from step 5 is freeze-dried under vacuum to obtain rice peptides.

[0012] Furthermore, in step 1, the crushed rice residue is sieved through a 60-mesh sieve.

[0013] Furthermore, in step 2, the organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3-4.

[0014] Furthermore, in step 3, the slurry concentration is 5%, the amount of α-amylase added is 1%, and the molecular weight cutoff of the nanofiltration membrane is <1000 Da.

[0015] Furthermore, in step 4, the specific operation of enzymatic hydrolysis is as follows:

[0016] ① First, add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, and time 1-2 hours;

[0017] ② Next, add neutral protease at a concentration of 2%, pH 6.0~7.0, temperature 40~50℃, and time 2-3 hours;

[0018] ③ Finally, add flavor protease at a rate of 1%, pH 6.0~7.0, temperature 45~55℃, and time 1-2 hours;

[0019] During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder is added simultaneously, with the mass ratio of CaCO3 powder to neutral protease and flavor protease being 0.5-0.8:1.

[0020] Furthermore, in step 5, the specific operation of membrane filtration is as follows:

[0021] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0022] ②Then, membrane filtration was performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate was collected.

[0023] Furthermore, in step 6, the molecular weight of the rice peptide is 200-1000 Da.

[0024] To achieve the above objectives, the present invention also provides a rice peptide that enhances immunity, which is prepared by the above preparation method.

[0025] Furthermore, the molecular weight of rice peptides is 200-1000 Da.

[0026] To achieve the above objectives, the present invention also provides the application of the above-mentioned rice peptides in the preparation of products that enhance immunity, wherein the products are health foods.

[0027] The advantages and positive effects of the method for preparing rice peptides that enhance immunity as described in this invention are:

[0028] 1. This invention effectively removes physical impurities and controls the particle size of raw materials through graded crushing and washing, providing a uniform substrate for subsequent enzymatic hydrolysis and separation, thus improving mass transfer efficiency and reaction uniformity. Simultaneously, the defatting process thoroughly removes fat from the rice residue, preventing fat oxidation and rancidity, and ensuring the flavor stability and shelf life of the product. Most importantly, it separates soluble sugars. First, α-amylase hydrolyzes starch, converting macromolecular starch into soluble small-molecule sugars. Then, a nanofiltration membrane with a density of <1000 Da is used to remove these sugars, avoiding browning during enzymatic hydrolysis. This also significantly increases the relative concentration of protein / peptide in the subsequent enzymatic hydrolysis system, allowing the protease to act more efficiently on the target substrate, reducing enzyme dosage, increasing yield, and avoiding excessive sugar interference in the final product.

[0029] 2. This invention employs a stepwise compound enzymatic hydrolysis process, sequentially using alkaline protease, neutral protease, and flavor protease for hydrolysis, resulting in more thorough hydrolysis and increased peptide yield; simultaneously, Ca is added during the hydrolysis process. 2+ Salt, Ca 2+ Salt can neutralize the H released during protease hydrolysis. + This stabilizes the pH of the reaction system, ensuring that the three proteases always operate efficiently near their respective optimal pH values; and most proteases are metalloenzymes, Ca... 2+ As an activator and stabilizer, it greatly improves enzyme activity and stability, reduces enzyme dosage, and shortens reaction time; it significantly increases enzymatic hydrolysis efficiency and degree of hydrolysis, achieving high yield of rice peptides.

[0030] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0031] The technical solution of the present invention will be further described below through embodiments.

[0032] Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0033] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0034] Alkaline protease (200,000 U / g), neutral protease (6,000 U / g), and flavor protease (500-2,000 U / g) were purchased from Sigma.

[0035] Example 1

[0036] A method for preparing rice peptides that enhance immunity includes the following steps:

[0037] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it 3 times with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0038] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0039] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3.

[0040] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry with a concentration of 5%. Add 1% α-amylase and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme. Adjust the slurry concentration to 10%, centrifuge and collect the supernatant. Use a nanofiltration membrane with a molecular weight cutoff of <1000 Da to separate soluble sugars and keep the retentate for later use.

[0041] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use.

[0042] The specific steps for enzymatic hydrolysis are as follows:

[0043] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0044] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0045] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0046] During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder is added simultaneously, with a mass ratio of CaCO3 powder to neutral protease and flavor protease of 0.5:1.

[0047] Step 5, membrane filtration purification:

[0048] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0049] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0050] Step 6, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0051] Example 2

[0052] A method for preparing rice peptides that enhance immunity includes the following steps:

[0053] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it twice with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0054] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0055] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:4.

[0056] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry with a concentration of 5%. Add 1% α-amylase and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme. Adjust the slurry concentration to 10%, centrifuge and collect the supernatant. Use a nanofiltration membrane with a molecular weight cutoff of <1000 Da to separate soluble sugars and keep the retentate for later use.

[0057] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 7%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use.

[0058] The specific steps for enzymatic hydrolysis are as follows:

[0059] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0060] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0061] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0062] During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder was added simultaneously, with a mass ratio of CaCO3 powder to neutral protease and flavor protease of 0.6:1.

[0063] Step 5, membrane filtration purification:

[0064] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0065] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0066] Step 6, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0067] Example 3

[0068] A method for preparing rice peptides that enhance immunity includes the following steps:

[0069] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it 3 times with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0070] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0071] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3.

[0072] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry with a concentration of 5%. Add 1% α-amylase and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme. Adjust the slurry concentration to 10%, centrifuge and collect the supernatant. Use a nanofiltration membrane with a molecular weight cutoff of <1000 Da to separate soluble sugars and keep the retentate for later use.

[0073] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 7%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use.

[0074] The specific steps for enzymatic hydrolysis are as follows:

[0075] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0076] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0077] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0078] During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder was added simultaneously, with a mass ratio of CaCO3 powder to neutral protease and flavor protease of 0.8:1.

[0079] Step 5, membrane filtration purification:

[0080] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0081] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0082] Step 6, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0083] Comparative Example 1

[0084] A method for preparing rice peptides that enhance immunity includes the following steps:

[0085] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it 3 times with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0086] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0087] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3.

[0088] Step 3, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use;

[0089] The specific steps for enzymatic hydrolysis are as follows:

[0090] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0091] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0092] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0093] During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder is added simultaneously, with a mass ratio of CaCO3 powder to neutral protease and flavor protease of 0.5:1.

[0094] Step 4, membrane filtration purification:

[0095] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0096] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0097] Step 5, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0098] Comparative Example 2

[0099] A method for preparing rice peptides that enhance immunity includes the following steps:

[0100] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it 3 times with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0101] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0102] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3.

[0103] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry with a concentration of 5%. Add 1% α-amylase and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme. Adjust the slurry concentration to 10%, centrifuge and collect the supernatant. Use a nanofiltration membrane with a molecular weight cutoff of <1000 Da to separate soluble sugars and keep the retentate for later use.

[0104] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use.

[0105] The specific steps for enzymatic hydrolysis are as follows:

[0106] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0107] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0108] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0109] Step 5, membrane filtration purification:

[0110] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0111] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0112] Step 6, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0113] Comparative Example 3

[0114] A method for preparing rice peptides that enhance immunity includes the following steps:

[0115] Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it 3 times with distilled water, dry it for later use, crush the dried rice residue and pass it through a 60-mesh sieve to obtain rice residue powder.

[0116] Step 2, Degreasing: Degreasing is carried out by organic solvent leaching. After degreasing, steam desolventizing is performed. After the solid is washed and dried, degreased rice residue powder is obtained.

[0117] The organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3.

[0118] Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry with a concentration of 5%. Add 1% α-amylase and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.5. After enzymatic hydrolysis, inactivate the enzyme. Adjust the slurry concentration to 10%, centrifuge and collect the supernatant. Use a nanofiltration membrane with a molecular weight cutoff of <1000 Da to separate soluble sugars and keep the retentate for later use.

[0119] Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15 minutes, and collect the supernatant for later use.

[0120] The specific steps for enzymatic hydrolysis are as follows:

[0121] ① Add alkaline protease at a concentration of 1%, pH 7.0~8.0, temperature 30~40℃, time 1h;

[0122] ② Add neutral protease at a concentration of 2%, maintain pH at 6.0-7.0, temperature at 40-50℃, and time at 2 hours;

[0123] ③ Add flavor protease, 1% addition amount, pH 6.0~7.0, temperature 45~55℃, time 1h;

[0124] During the enzymatic hydrolysis of neutral protease and flavor protease, NaCl powder was added simultaneously, with a mass ratio of NaCl powder to neutral protease and flavor protease of 0.5:1.

[0125] Step 5, membrane filtration purification:

[0126] ① First, membrane filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate is collected;

[0127] ②Then, membrane filtration is performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate is collected;

[0128] Step 6, Drying: The retentate from Step 5 is freeze-dried under vacuum to obtain rice peptides with a molecular weight of 200-1000 Da.

[0129] Effect verification:

[0130] Browning degree determination: Dissolve 1g of rice peptide in 5mL of distilled water, using distilled water as a control, and measure the absorbance (A) at 420nm. 420 Each sample was repeated 3-4 times, and the average value was taken.

[0131] Color difference determination: Dissolve 1g of rice peptide in 5mL of distilled water, place the rice peptide sample in a cuvette, and measure the color difference using a colorimeter. L , a and b Before testing, the colorimeter was calibrated using a standard white board. L The lightness of the sample; a The value represents the redness of the sample; a positive value indicates a reddish tint, while a negative value indicates a greenish tint. b The value represents the yellowness of the sample; a positive value indicates a yellowish tint, and a negative value indicates a bluish tint. Each sample is tested in 6-8 replicates, and the average value is taken. The color difference of distilled water is used as the blank. ΔE ) and saturation C The calculation formula is as follows:

[0132] Total color difference ΔE = ;

[0133] Saturation C = .

[0134] The browning degree and color difference value of the rice peptides from Examples 1-3 and Comparative Examples 1-3 were measured, and the results are shown in Table 1:

[0135] Table 1. Browning Degree and Color Difference Values

[0136]

[0137] As shown in Table 1, Comparative Examples 1-3 ΔE The value is significantly greater than that of Examples 1-3. ΔEThe value indicates that the overall color deviation of the comparative sample is greater compared to the colorless state of pure water. This suggests that the comparative sample is darker in color, deviates further from colorless, and is of lower quality. All samples... L The values ​​are all far below 100, indicating that they are significantly darker than pure water. However, in the examples... L The value is still significantly higher than the comparative value. L Value. This indicates that the example samples are brighter and whiter than the control samples. All samples have a high value. b Value and C The values ​​indicate that they all exhibit a distinct yellow hue. However, the proportions... b Value and C The value is higher than that of the comparative example. This indicates that the comparative sample is more yellow and has a more intense color (higher saturation), suggesting that the browning in the comparative example is more severe. (Comparative Example) a The value is negative, with a slight green tint. (Comparative analysis) a The value is positive, slightly reddish. Browning usually results in a reddish-brown color in the product, indicating that the browning in the comparative example is more severe. The A420 value of the comparative example is higher than that of the example. This is completely consistent with the conclusion of the colorimetric data, directly proving that the browning in the comparative example is more severe. Therefore, the rice peptides of Examples 1, 2, and 3 are significantly better in color than those of Comparative Examples 1, 2, and 3. Specifically, the color is lighter and brighter, the yellow is paler, and the degree of browning is less. This indicates that the soluble sugar separation step and the enzymatic hydrolysis with the addition of CaCO3 reduced the browning of the rice peptides, thus improving their quality.

[0138] Immunity enhancement assay:

[0139] 1g of rice peptide was dissolved in 5mL of distilled water to prepare a rice peptide solution. The experimental subjects were inbred female mice, weighing 18-22g, with 10-15 mice in each group, divided into 4 groups. The grouping and drug administration are shown in Table 2.

[0140] Table 2 Grouping and Dosing Information

[0141]

[0142] Thirty days later, mice in each group underwent an experiment on the phagocytosis of chicken red blood cells by mouse peritoneal macrophages.

[0143] Utilizing the adhesion property of macrophages to smooth surfaces such as glass, peritoneal fluid containing macrophages was dropped onto a glass slide, chicken erythrocytes were added, and after incubation for a certain period, the non-adhering cells were washed away, the slide was fixed and stained, and the phagocytic rate and phagocytic index of macrophages that had engulfed chicken erythrocytes were counted under a microscope to determine the phagocytic capacity of macrophages. This principle was applied to determine the phagocytic capacity of mouse macrophages.

[0144] a. Instruments and materials:

[0145] Microscope, 37℃ incubator, counter, a set of surgical instruments, syringe, dropper, rubber-tipped pipette, bulb syringe, glass slide, staining tank, test tube.

[0146] ① Slide preparation:

[0147] Reusable slides should be soaked in washing solution, rinsed, and air-dried, then soaked in alcohol overnight. Before use, wipe dry with gauze or air-dry; otherwise, it will affect macrophage adhesion and microscopic examination. Label the slides, and draw two circles on each slide with 3% agar (the circles must be completely sealed to prevent liquid leakage), then air-dry for later use.

[0148] ② Enamel or plastic box: Line the inside with semi-damp gauze (wet it thoroughly with warm water and place it in the incubator for later use), and make sure the gauze is flat.

[0149] ③ Reagent preparation method

[0150] Preparation of agar: Take 3g of agar, add 100mL of water, heat to boiling until transparent, and add 1-2 drops of 1% bromocresol purple indicator.

[0151] Preparation method of PBS buffer: Dissolve 6.66g KH2PO4 and 6.38g Na2HPO4•12H2O in 1000mL of distilled water and adjust the pH to 7.2.

[0152] Chicken red blood cell suspension:

[0153] Before the experiment, collect blood from the jugular vein or artery of chickens and place it in an Erlenmeyer flask containing about 20 glass beads. Shake thoroughly in one direction for 5-10 minutes to remove fibrin and store at 4°C. Wash the blood three times with physiological saline before the experiment, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and prepare a 1% red blood cell suspension using Hank's solution according to hematocrit.

[0154] Giemsa stain:

[0155] I. Take 0.5g of Giemsa dye, 33mL of neutral glycerol, and 33mL of methanol. First, place the Giemsa dye in a clean mortar, add glycerol, grind briefly, pour into a brown bottle, place in a water bath at 55-60℃ for 2 hours, shaking constantly, then add methanol and shake well. Store for later use.

[0156] II. Diluting Giemsa staining solution: When using, add 1 part of Giemsa staining solution stock solution to 8 parts of pH 6.8 buffer solution to prepare the working solution.

[0157] Giemsa stain decolorizing solution: Preparation method: Mix 20 mL methanol and 80 mL distilled water, then add 2 drops of 2N HCl.

[0158] b. Experimental steps:

[0159] ① Activation of mouse macrophages: Four days before the experiment, each mouse was injected intraperitoneally with 0.2 mL of 2% hematocrit sheep red blood cells. Mice were euthanized by cervical dislocation, and 4 mL of Hank's solution with fetal bovine serum was injected intraperitoneally per mouse. The abdomen was gently massaged 20 times to thoroughly wash out peritoneal macrophages. Then, a small incision was made in the abdominal wall, and 2 mL of peritoneal lavage fluid was drawn into a test tube using a pipette (or a syringe).

[0160] ② Using a 1mL pipette, add 0.5mL of peritoneal lavage fluid to a test tube containing 0.5mL of 1% chicken blood erythrocyte suspension and mix well. Using a syringe (with a large needle), add 0.5mL of the mixture to an agar ring on a glass slide. Incubate at 37℃ for 15-20 minutes. After incubation, quickly rinse away any non-adherent cells with physiological saline, fix in methanol for 1 minute, and stain with Giemsa solution for 15 minutes. Rinse thoroughly with distilled water, air dry, and count the phagocytic rate and phagocytic index using a 40× microscope. The phagocytic rate is the percentage of macrophages that phagocytose chicken erythrocytes per 100 macrophages; the phagocytic index is the average number of chicken erythrocytes phagocytosed by each macrophage. The experimental results are shown in Table 3.

[0161] Table 3. Phagocytosis rate and phagocytosis index (mean)

[0162]

[0163] c. Data processing and result determination:

[0164] For a test result to be considered positive, the phagocytic percentage or phagocytic index of the tested sample group must show a significant difference compared to the control group. Table 3 shows that the phagocytic rate and phagocytic index of the mice in Examples 1-3 were significantly higher than those in the blank control group, indicating that the phagocytic capacity of the phagocytes in the mice in Examples 1-3 was strong and their immunity was significantly improved. This demonstrates that the rice peptides prepared in Examples 1-3 can improve the immunity of mice, thus indicating that rice peptides can be used to prepare related products that enhance immunity.

[0165] Therefore, the present invention adopts the above-mentioned method for preparing rice peptides that enhance immunity. By reducing the content of reducing sugars, inhibiting browning, and improving the quality of rice peptides, the prepared rice peptides have the effect of enhancing immunity.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing rice peptides that enhance immunity, characterized in that, Includes the following steps: Step 1, sorting and crushing: Remove impurities from the rice residue, use a vibrating screen to screen the rice residue with a mesh size of 20-40, then wash it with distilled water 2-3 times, dry it for later use, crush the dried rice residue and pass it through a sieve of 60-80 mesh to obtain rice residue powder. Step 2, Degreasing: The rice residue powder is degreased by organic solvent leaching. After degreasing, it is desolventized by steam. The solid is washed and dried to obtain degreased rice residue powder. Step 3, soluble sugar separation: Add defatted rice residue powder to deionized water to prepare a slurry, add α-amylase, and enzymatically hydrolyze the starch at 60-70℃ and pH 6.0-6.

5. After enzymatic hydrolysis, inactivate the enzyme; adjust the slurry concentration to 10%, centrifuge and collect the supernatant, use a nanofiltration membrane to separate soluble sugars, and collect the retentate for later use; The slurry concentration was 5%, the amount of α-amylase added was 1%, and the molecular weight cutoff of the nanofiltration membrane was <1000 Da. Step 4, enzymatic hydrolysis: Add distilled water to the retentate to adjust the substrate concentration to 6-8%, carry out enzymatic hydrolysis, inactivate the enzyme after the enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysate at 5000-8000 rpm for 15-20 minutes, and collect the supernatant for later use. The specific steps of enzymatic hydrolysis are as follows: ① First, add alkaline protease at a concentration of 1%, pH 7.0–8.0, temperature 30–40℃, and time 1–2 hours; ② Next, add neutral protease at a dosage of 2%, pH 6.0-7.0, temperature 40-50℃, and time 2-3 hours; ③ Finally, add flavor protease at a dosage of 1%, pH 6.0-7.0, temperature 45-55℃, and time 1-2 hours; During the enzymatic hydrolysis of neutral protease and flavor protease, CaCO3 powder is added simultaneously, with the mass ratio of CaCO3 powder to neutral protease and flavor protease being 0.5-0.8:

1. Step 5, membrane filtration purification: membrane filtration is performed sequentially using ultrafiltration membrane and nanofiltration membrane, and finally the retentate is collected; Step 6, Drying: The retentate from step 5 is freeze-dried under vacuum to obtain rice peptides.

2. The method for preparing an immune-enhancing rice peptide according to claim 1, characterized in that: In step 1, the crushed rice residue is sieved through a 60-mesh sieve.

3. The method for preparing an immune-enhancing rice peptide according to claim 1, characterized in that, In step 2, the organic solvent is n-hexane, and the degreasing conditions are: temperature 50-60℃, time 1-2h, and liquid-to-solid ratio w / v of 1:3-4.

4. The method for preparing an immune-enhancing rice peptide according to claim 1, characterized in that, In step 5, the specific operation of membrane filtration is as follows: ① First, use an ultrafiltration membrane with a molecular weight cutoff of 5000 Da for membrane filtration and collect the permeate; ②Then, membrane filtration was performed using a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, and the retentate was collected.

5. The method for preparing an immune-enhancing rice peptide according to claim 1, characterized in that: In step 6, the molecular weight of rice peptides is 200-1000 Da.

6. A rice peptide that enhances immunity, characterized in that: It is prepared by the preparation method according to any one of claims 1-5.

7. The rice peptide for enhancing immunity according to claim 6, characterized in that: The molecular weight of rice peptides is 200-1000 Da.

8. The application of the rice peptide for enhancing immunity as described in claim 6 or 7 in the preparation of products for enhancing immunity, characterized in that: The product in question is a health food product.

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