A red kidney bean bacteriostatic active polypeptide and a preparation method thereof

Abstract

The present application relates to a kind of red kidney bean bacteriostatic active polypeptide and its preparation method.The preparation method includes the following preparation steps: S100, raw material pretreatment: the red kidney bean dreg of extracting albumin is softened and grinded homogenization to be made into slurry by adding water;S200, hydrolysis: two kinds of neutral protease hydrolysis are added to the slurry, to obtain hydrolysate;S300, membrane separation: the hydrolysate is inactivated after enzyme, and solid-liquid separation is carried out, and the obtained filtrate is separated using the ultrafiltration membrane with the molecular weight cut-off 3000Da, and the obtained ultrafiltrate is desalted and concentrated using the nanofiltration membrane with the molecular weight cut-off 300Da, to obtain red kidney bean bacteriostatic active polypeptide concentrate.The polypeptide prepared by the method of the present application has inhibitory effect on streptococcus mutans, salmonella and staphylococcus aureus.Because these bacteria are common digestive tract pathogenic bacteria, the polypeptide can be used as a product for inhibiting harmful bacteria in digestive tract or as an external bacteriostatic product.

Description

Technical Field

[0001] This invention relates to the technical field of red kidney bean polypeptides, and particularly to a red kidney bean antibacterial active polypeptide and its preparation method. Background Technology

[0002] Red kidney bean (Phaseolus vulgaris Linn.sp.) is the seed of a plant in the genus Phaseolus L. In China, the most common varieties cultivated are large and small red kidney beans. Red kidney beans contain 22%–24% protein, including water-soluble albumin, which is an α-amylase inhibitor.

[0003] Previous studies have shown that after extracting water-soluble albumin from red kidney beans, the residue still contains 6% to 12% water-insoluble protein. If these residual proteins can be processed into active polypeptides, the economic value and comprehensive utilization value of deep processing of red kidney beans can be improved, and the development of red kidney bean cultivation can be promoted.

[0004] Currently, there are few research papers and patents on kidney bean peptides. Dong Xianhui reported in her article "Research on the Formulation of Chewable Tablets Containing Antioxidant Peptides from Red Kidney Bean Protein" that red kidney bean antioxidant peptides prepared using alkaline protease can be processed into chewable tablets. Zhang Jing, in her article "Research on the Extraction Process of Kidney Bean Peptides," also used alkaline protease to process kidney bean peptides. Patent document CN202010861723.6, "A Kidney Bean Bioactive Peptide and Its Application in the Preparation of Uric Acid-Lowering Drugs," discloses the extraction of kidney bean protein using an alkaline dissolution and acid precipitation method, followed by the preparation of peptides with xanthine oxidase inhibitory activity using alkaline protease. Patent ZL201410352633.9... The patent document "A Functional White Kidney Bean Polypeptide and Its Preparation Method and Application" mentions that enzymatic hydrolysis of white kidney bean albumin with acidic protease can yield a polypeptide with high thermal stability, which has the activity of inhibiting α-amylase. Wang Qiuming reported in the article "Research and Development of White Kidney Bean Polypeptides and Amino Acid Umami Agents" that, using soybean residue from which white kidney bean albumin was extracted as raw material, the protein was first extracted and separated by alkaline dissolution and acid precipitation. Then, the effects of hydrolyzing the separated protein with plant protein hydrolysis enzymes, papain, animal protein hydrolysis complex enzymes and alkaline protease were compared to prepare polypeptides and amino acids. The results showed that the hydrolysis effect of alkaline protease was the best, and the yield of polypeptides and amino acids could reach up to 5.67%.

[0005] There are currently no reports of using red kidney beans to prepare antibacterial active peptides. Summary of the Invention

[0006] To address the problems mentioned in the background section, this invention provides a method for preparing antibacterial active peptides from red kidney beans, comprising the following preparation steps:

[0007] S100. Raw material pretreatment: The red kidney bean residue after extracting albumin is softened with water and ground into a homogenized pulp.

[0008] S200, Hydrolysis: Add Aspergillus melleus neutral protease and Bacillus subtilis neutral protease to the slurry for hydrolysis to obtain hydrolysate;

[0009] S300, Membrane separation: After inactivating the enzyme in the hydrolysate, solid-liquid separation is performed to obtain a filtrate. The filtrate is then separated using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The obtained ultrafiltrate is then desalted and concentrated using a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain the concentrated solution of the red kidney bean antibacterial active polypeptide.

[0010] In one embodiment, the preparation method of the slurry is as follows: red kidney bean residue from which albumin has been extracted is mixed with water at a mass ratio of 1:10-20 and soaked at 10-20°C for 6-12 hours; then the mixture is ground at least once with a colloid mill, and 2%-3% of edible salt by mass of the ground slurry is added and dissolved. The mixture is then heated to 55-65°C and kept warm for 40-80 minutes, and homogenized at least once under a pressure of 5-10 MPa to obtain the slurry.

[0011] Adding a certain amount of salt to the grinding slurry can promote the dissolution of globulin in red kidney bean residue and raise the gelatinization temperature of red kidney bean starch to above 67℃. This is beneficial for extracting globulin from the homogenized slurry at 55-65℃ without gelatinizing the starch, and makes the polypeptide hydrolysate easier to separate.

[0012] In one embodiment, during the hydrolysis process, the amount of enzyme added is based on the protein content in the slurry; the amount of Aspergillus honeysuckle neutral protease added is 3000-5000 U / g; and the amount of Bacillus subtilis neutral protease added is 1500-3000 U / g.

[0013] In one embodiment, the Aspergillus honeysuckle neutral protease may be the 3SD type protease from Amano Corporation, but is not limited thereto; the Bacillus subtilis neutral protease may be the 1398 strain from Nanning Pangbo Biotechnology Co., Ltd., but is not limited thereto.

[0014] In one embodiment, the hydrolysis conditions are 40–50°C, pH 7.0–8.5, and hydrolysis for 90–160 min.

[0015] In one embodiment, the pH of the hydrolysate is adjusted to 4-5 before enzyme inactivation.

[0016] In one embodiment, the enzyme inactivation condition is to heat the hydrolysate to boiling for 4 to 8 minutes.

[0017] In one embodiment, the filtrate is obtained by first allowing the enzyme to be inactivated by hydrolysis solution to stand for 24-48 hours, and then taking the supernatant and filtering it through a 50nm ceramic membrane.

[0018] In one embodiment, the red kidney bean antibacterial active polypeptide concentrate contains sodium chloride content not exceeding 2% of the soluble solids content.

[0019] In one embodiment, the concentrated red kidney bean antibacterial active polypeptide solution can also be spray-dried into powder for preservation.

[0020] The present invention also provides a red kidney bean antibacterial active polypeptide, which is prepared by any of the above-described methods for preparing red kidney bean antibacterial active polypeptides.

[0021] Based on the above, compared with the prior art, the method for preparing a red kidney bean antibacterial active polypeptide provided by the present invention has the following beneficial effects:

[0022] 1. Compared with the previously reported process for preparing polypeptides from red kidney bean residue, this invention eliminates the need for alkali dissolution and acid precipitation to extract proteins, and directly adds enzyme hydrolysis, simplifying the process and reducing wastewater discharge. According to testing, this invention can achieve a polypeptide extraction rate of 6.2% from red kidney bean residue.

[0023] 2. This invention utilizes a dual-enzyme approach—using Aspergillus oryzae neutral protease and Bacillus subtilis neutral protease—to simultaneously hydrolyze red kidney bean residue. The hydrolysis time is short, the process is simple, and the resulting polypeptide exhibits inhibitory activity against Streptococcus mutans, Salmonella, and Staphylococcus aureus. Streptococcus mutans is a major pathogenic bacterium causing dental caries, while Salmonella and Staphylococcus aureus are common intestinal pathogens. Therefore, this polypeptide can be used as a product to inhibit harmful bacteria in the digestive tract or as a topical antibacterial product. Since the polypeptide raw material is derived from food, it has good safety and can also be used as a protein food ingredient.

[0024] Other features and beneficial effects of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects of the invention and other beneficial effects may be realized and obtained by means of the structures and / or components pointed out in the description and claims. Attached Figure Description

[0025] Figure 1 This image shows the antibacterial effect of red kidney bean peptides on Streptococcus mutans.

[0026] Figure 2 This image shows the antibacterial effect of red kidney bean peptides on Salmonella.

[0027] Figure 3 This image shows the antibacterial effect of red kidney bean polypeptide against Staphylococcus aureus.

[0028] Figure 4The image shows the antibacterial concentration gradient curve of the red kidney bean polypeptide sample in Example 2. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0031] The present invention provides the following embodiments.

[0032] Example 1

[0033] S100. Raw material pretreatment: Weigh 100g of dried red kidney bean residue (protein content of 12.3%) that has been extracted with albumin, add 1700g of purified water, soak at 10℃ for 12 hours, grind into a slurry once with a colloid mill, add 36g of edible salt to dissolve, heat to 55℃ and keep warm for 80 minutes, homogenize once under high pressure at 5MPa to form a homogenized red kidney bean slurry.

[0034] S200, Hydrolysis: Adjust the pH of the homogenized slurry to 7.2, add 40,000 U of Aspergillus oryzae neutral protease and 36,000 U of Bacillus subtilis neutral protease, and hydrolyze at 40℃ for 160 min to obtain hydrolysate.

[0035] S300, Membrane Separation: Adjust the pH of the hydrolysate to 4.2, boil to inactivate enzymes for 4 minutes, cool and stand for 2 days, filter the supernatant with a 50nm ceramic membrane, separate the filtrate with an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then nanofiltration and concentration with a 300Da nanofiltration membrane until the sodium chloride content is less than 2% of the soluble solids content in the solution. Spray dry the polypeptide concentrate to obtain red kidney bean polypeptide powder.

[0036] Analysis showed that the polypeptide obtained in this example weighed 6.22 grams, with a relative yield of 6.22% of the raw materials.

[0037] The red kidney bean polypeptide powder was prepared into a solution with a polypeptide content of 20 mg / mL for antibacterial testing. The bacteria used in the antibacterial test were Streptococcus mutans, Salmonella, and Staphylococcus aureus. The specific test method was as follows:

[0038] Heat the nutrient agar medium to a boil, then cool it to 50–60°C and pour it into three petri dishes. After solidification, add 10 μg of the culture medium to each dish. 4 The three bacterial suspensions were evenly spread onto the surfaces of three culture media. Small holes with a diameter of 3-3.5 mm were punched on the surface of the culture media, and 40 μL of polypeptide at a concentration of 20 mg / ml was added to the small holes. The media were then incubated at 37°C for 48 h, and the size of the inhibition zone was observed.

[0039] Figure 1 This image shows the antibacterial effect of red kidney bean peptides against Streptococcus mutans. Figure 1 Red kidney bean polypeptide solution was injected into wells A and B, and distilled water was injected into wells C and D. The inhibition zone diameter of Streptococcus mutans in wells A and B was about 8.3 mm.

[0040] Figure 2 This image shows the antibacterial effect of red kidney bean peptides against Salmonella. Figure 2 Red kidney bean polypeptide solution was injected into H and F wells, and distilled water was injected into E and G wells. The inhibition zone diameter of Salmonella in H and F wells was about 8.0 mm.

[0041] Figure 3 This image shows the antibacterial effect of red kidney bean polypeptide against Staphylococcus aureus. Figure 3 Red kidney bean polypeptide solution was injected into the R and S wells, and distilled water was injected into the T and U wells. The inhibition zone diameter of Staphylococcus aureus in the R and S wells was about 10.3 mm.

[0042] from Figures 1 to 3 As shown in the antibacterial effect diagram, the red kidney bean polypeptide prepared in this invention has a significant inhibitory effect on the above-mentioned pathogenic bacteria.

[0043] Example 2

[0044] S100. Raw material pretreatment: Weigh 100g of red kidney bean wet residue (protein content of 5.8%) after extracting albumin, add 1000g of softened water, soak at 20℃ for 6 hours, grind into a slurry using a colloid mill, add 30g of edible salt to dissolve in the slurry, heat to 60℃ and keep warm for 60 minutes, homogenize once at 10MPa using a homogenizer to form a homogenized red kidney bean slurry.

[0045] S200, hydrolysis: Adjust the pH of the homogenized slurry to 8.3, add 25,000 U of Aspergillus oryzae neutral protease and 11,000 U of Bacillus subtilis neutral protease, and hydrolyze at 50℃ for 90 min to obtain hydrolysate.

[0046] S300, Membrane Separation: Adjust the pH of the hydrolysate to 4.7, boil to inactivate enzymes for 8 minutes, cool and stand for 36 hours, filter the supernatant with a 50nm ceramic membrane, and then use a 3000Da ultrafiltration membrane for ultrafiltration separation. The ultrafiltrate is then nanofiltration and concentrated using a 300Da nanofiltration membrane. When the sodium chloride content in the nanofiltration retentate drops to 1.8% of the soluble solids content in the solution, the polypeptide solution is spray-dried to obtain red kidney bean polypeptide powder.

[0047] Analysis showed that the peptide yield relative to the raw materials in this embodiment was 3.57%.

[0048] The antibacterial effects of red kidney bean polypeptides at concentrations of 0, 5, 10, 20, 30, 40, and 50 mg / ml against Streptococcus mutans, Salmonella, and Staphylococcus aureus were tested using nutrient agar medium. The culture and observation methods were the same as in Example 1.

[0049] From the concentration inhibition curve (see) Figure 4 It can be seen that the red kidney bean polypeptide has a better inhibitory effect on Streptococcus mutans and Salmonella at a concentration of 10-35 mg / ml; and a better inhibitory effect on Staphylococcus aureus at a concentration of 10-45 mg / ml.

[0050] Since the red kidney bean antibacterial active peptide provided by this invention is a mixture, it contains not only the red kidney bean antibacterial active peptide but also some nutrients such as soluble starch or sugar. Therefore, the antibacterial effect of this red kidney bean antibacterial active peptide is not completely linearly positively correlated with the peptide concentration.

[0051] Example 3

[0052] S100. Raw material pretreatment: Take 100g of crushed red kidney bean residue (protein content of 10.6%), add 1600g of purified water, soak at 15℃ for 8 hours, grind into a slurry once with a colloid mill, add 46g of edible salt to dissolve, heat to 65℃ and keep warm for 40 minutes, homogenize once at 8MPa to form a homogenized red kidney bean slurry.

[0053] S200, Hydrolysis: Adjust the pH of the homogenized slurry to 7.8, add 50,000 U of Aspergillus oryzae neutral protease and 20,000 U of Bacillus subtilis neutral protease, and hydrolyze at 45℃ for 120 min to obtain hydrolysate.

[0054] S300, Membrane Separation: Adjust the pH of the hydrolysate to 4.5, boil to inactivate enzymes for 6 minutes, then cool and stand for 40 hours. Filter the supernatant through a 50nm ceramic membrane to obtain the filtrate. The relative yield of peptides in the filtrate was determined to be 5.93%.

[0055] The filtrate was separated by ultrafiltration using ultrafiltration membranes with pore sizes of 10000 Da, 5000 Da, 3000 Da, and 1000 Da, respectively, to obtain ultrafiltrates with Mw values ​​of 10000–5000 Da, 5000–3000 Da, <3000 Da, and <1000 Da.

[0056] The concentrations of the peptides with different molecular weights were adjusted to 20 mg / ml, and then the antibacterial effects of the peptide solutions with different molecular weights were tested. The bacteria tested were Staphylococcus aureus and Salmonella, and the culture medium was nutrient agar. The operation, culture, and observation methods were the same as in Example 1.

[0057] The test results are shown in Table 1:

[0058] Table 1 - Antibacterial effects of different molecular weight peptide fragments (20 mg / ml)

[0059] 5000Da-10000Da 3000Da-5000Da <3000Da <1000Da 300Da-3000Da Staphylococcus aureus inhibition zone (cm) - 3.8 9.8 6.0 10.7 inhibition zone of Salmonella (cm) - - 7.5 5.2 8.5

[0060] As shown in Table 1, peptides with a molecular weight <3000 Da and Mw <1000 Da both exhibited antibacterial effects in the samples prepared in this embodiment. However, the antibacterial effect of peptides with Mw <1000 Da was weaker than that of peptides with Mw <3000 Da. Therefore, peptides with a molecular weight below 3000 Da were selected as the antibacterial products of this invention. Furthermore, in this embodiment, a peptide solution with a molecular weight below 3000 Da was separately subjected to 300 Da nanofiltration to achieve a sodium chloride content of 1.5% of the soluble solids content in the nanofiltration retentate. After adjusting the nanofiltration peptide to 20 mg / ml, an inhibition zone experiment was conducted. The results showed that the inhibition zones for Staphylococcus aureus and Salmonella were 10.7 cm and 8.5 cm, respectively, with the antibacterial effect being approximately 9%–13% higher than that of the unfiltered peptide. This may be because during nanofiltration desalination, small molecule nutrients such as monosaccharides in the hydrolysate are also filtered out through the nanofiltration membrane under pressure. Therefore, the amount of antimicrobial peptides interfering with the solution is reduced or the amount of nutrients available to microorganisms is reduced, thereby improving the antimicrobial effect.

[0061] Example 4

[0062] S100. Raw material pretreatment: Take 100g of crushed red kidney bean residue (protein content of 10.6%), add 1800g of purified water, soak at 10℃ for 12 hours, grind into a slurry using a colloid mill, add 54g of edible salt to the slurry to dissolve, heat to 60℃ and keep warm for 70 minutes, homogenize once under high pressure at 10MPa to form a homogenized red kidney bean slurry.

[0063] S200, Hydrolysis: Adjust the pH of the homogenized red kidney bean slurry to 7.5, add 38,000 U of Aspergillus honeysuckle neutral protease and 30,000 U / g of Bacillus subtilis neutral protease, and hydrolyze at 45℃ for 130 min to obtain hydrolysate.

[0064] S300, Membrane Separation: Adjust the pH of the hydrolysate to 4.5, boil to inactivate enzymes for 8 minutes, cool and stand for 28 hours, take the supernatant, filter with a 50nm ceramic membrane, separate the filtrate with a 3000Da ultrafiltration membrane, desalinate and concentrate the ultrafiltrate with a 300Da nanofiltration membrane, and spray dry to obtain red kidney bean polypeptide powder when the sodium chloride content in the retentate drops to 1.9% of the total soluble solids in the solution.

[0065] The present invention also provides the following comparative examples:

[0066] Comparative Example 1

[0067] During the hydrolysis process, only Aspergillus oryzae neutral protease was added, and Bacillus subtilis neutral protease was not added. Other operations and conditions were the same as in Example 4.

[0068] Comparative Example 2

[0069] During the hydrolysis process, only Bacillus subtilis neutral protease was added, and Aspergillus honeysuckle neutral protease was not added. Other operations and conditions were the same as in Example 4.

[0070] Comparative Example 3

[0071] During the hydrolysis process, the neutral protease of Bacillus subtilis was replaced with the neutral protease of Bacillus amyloliticus, and the hydrolysis conditions were changed to hydrolysis at 50°C for 130 min, with other conditions the same as in Example 4.

[0072] Comparative Example 4

[0073] During the hydrolysis process, the neutral protease of Aspergillus oryzae was replaced with the neutral protease of Bacillus amyloliticus, and the hydrolysis conditions were changed to hydrolysis at 50°C for 130 min, with other conditions the same as in Example 4.

[0074] Comparative Example 5

[0075] During the hydrolysis process, the neutral protease of Bacillus subtilis was replaced with enzymes such as Aspergillus oryzae neutral protease, and other conditions were the same as in Example 4.

[0076] Comparative Example 6

[0077] During the hydrolysis process, the neutral protease from Aspergillus oryzae was replaced with the neutral protease from Aspergillus honeysuckle, and other hydrolysis conditions were the same as in Example 4.

[0078] The red kidney bean polypeptide powders prepared in this embodiment and the above comparative example were prepared into a solution with a polypeptide content of 15 mg / ml, and their antibacterial effects against Streptococcus mutans, Salmonella and Staphylococcus aureus were tested. The culture and observation methods were the same as in Example 1, and the statistics of inhibition zones are shown in Table 2.

[0079] Table 2 - Inhibition zones of peptides hydrolyzed by different protease combinations

[0080] Streptococcus mutans (cm) inhibition zone of Salmonella (cm) Staphylococcus aureus inhibition zone (cm) Example 4 7.2 6.8 8.5 Comparative Example 1 none 5.5 6.4 Comparative Example 2 none none none Comparative Example 3 5.0 none none Comparative Example 4 none none none Comparative Example 5 none none 5.7 Comparative Example 6 none none none

[0081] The above experiments show that Comparative Example 2, which uses only peptides hydrolyzed by Bacillus subtilis neutral protease, and Comparative Examples 4 and 6, which replace Aspergillus honeysuckle neutral protease with Bacillus amyloliquefaciens neutral protease and Aspergillus oryzae neutral protease respectively, have no antibacterial effect.

[0082] Comparative Example 1, which was hydrolyzed solely by Aspergillus honey neutral protease, showed no inhibitory effect on Streptococcus mutans, and its antibacterial effect against Salmonella and Staphylococcus aureus was also inferior to that in Example 4, indicating that the antibacterial effect of kidney bean polypeptides hydrolyzed solely by Aspergillus honey neutral protease was poor.

[0083] Comparative Examples 3 and 5 replaced Bacillus subtilis neutral protease with Bacillus amyloliquefaciens neutral protease and Aspergillus oryzae neutral protease, respectively. The hydrolyzed peptides showed inhibitory activity against only a few pathogenic bacteria and had poor broad-spectrum activity.

[0084] Only Example 4, which uses the simultaneous compound hydrolysis of red kidney bean residue by Aspergillus honeysuckle neutral protease and Bacillus subtilis neutral protease provided by this invention, yields red kidney bean polypeptides with the best antibacterial effect.

[0085] In summary, this invention utilizes a dual-enzyme approach—using Aspergillus oryzae neutral protease and Bacillus subtilis neutral protease—to simultaneously hydrolyze red kidney bean residue. This method results in a short hydrolysis time, a simple process, and the obtained polypeptide exhibits inhibitory activity against Streptococcus mutans, Salmonella, and Staphylococcus aureus. Streptococcus mutans is a major pathogenic bacterium of dental caries, while Salmonella and Staphylococcus aureus are common intestinal pathogens. Therefore, this polypeptide can be used as a product to inhibit harmful bacteria in the digestive tract or as a topical antibacterial product. Since the polypeptide raw material is derived from food, it has good safety and can also be used as a protein food ingredient.

[0086] Furthermore, those skilled in the art should understand that although many problems exist in the prior art, each embodiment or technical solution of the present invention can be improved in only one or a few aspects, without necessarily solving all the technical problems listed in the prior art or the background art simultaneously. Those skilled in the art should understand that any content not mentioned in a claim should not be construed as a limitation on that claim.

[0087] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an antibacterial active polypeptide from red kidney beans, characterized in that: S100. Raw material pretreatment: The red kidney bean residue after extracting albumin is softened with water and ground into a homogenized slurry. S200, Hydrolysis: Add Aspergillus oryzae neutral protease and Bacillus subtilis neutral protease to the slurry for hydrolysis to obtain hydrolysate; S300, Membrane separation: After inactivating the enzyme in the hydrolysate, solid-liquid separation is performed to obtain a filtrate. The filtrate is then separated using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The obtained ultrafiltrate is then desalted and concentrated using a nanofiltration membrane with a molecular weight cutoff of 300 Da to obtain a concentrated solution of red kidney bean antibacterial active polypeptide.

2. The method for preparing the antibacterial active polypeptide from red kidney bean according to claim 1, characterized in that: The preparation method of the slurry is as follows: red kidney bean residue from which albumin has been extracted is mixed with water at a mass ratio of 1:10-20 and soaked at 10-20℃ for 6-12 hours; then the mixture is ground at least once with a colloid mill, and 2%-3% of edible salt is added to the slurry by mass to dissolve it. The mixture is then heated to 55-65℃ and kept warm for 40-80 minutes. The mixture is then homogenized at least once under a pressure of 5-10 MPa to obtain the slurry.

3. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 1, characterized in that: Add Aspergillus honeysuckle neutral protease and Bacillus subtilis neutral protease to the slurry, with dosages of 3000-5000 U / g and 1500-3000 U / g respectively, based on the crude protein content of the slurry.

4. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 1, characterized in that: The hydrolysis conditions are 40–50°C, pH 7.0–8.5, and hydrolysis for 90–160 min.

5. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 1, characterized in that: The pH of the hydrolysate is adjusted to 4-5 before enzyme inactivation.

6. The method for preparing the antibacterial active polypeptide from red kidney bean according to claim 1, characterized in that: The enzyme inactivation conditions are to heat the hydrolysate to boiling for 4–8 minutes.

7. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 1, characterized in that: The concentrated antibacterial active polypeptide contains sodium chloride at a content not exceeding 2% of the soluble solids content.

8. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 7, characterized in that: The concentrated antibacterial active polypeptide solution from red kidney beans can also be spray-dried into powder for preservation.

9. The method for preparing the antibacterial active polypeptide from red kidney beans according to claim 1, characterized in that: The filtrate is obtained by hydrolyzing the enzyme-inactivated solution, allowing it to stand for 24–48 hours, and then filtering the supernatant through a 50 nm ceramic membrane.

10. A red kidney bean antibacterial active polypeptide, characterized in that: The red kidney bean antibacterial active polypeptide was prepared by the method described in any one of claims 1 to 9.

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