A chicken gizzard membrane active polypeptide, its preparation method, and functional products

Abstract

This invention relates to the field of chicken gizzard lining processing technology, and particularly to an active polypeptide of chicken gizzard lining, its preparation method, and functional products. The preparation method includes the following steps: mixing chicken gizzard lining powder with water and subjecting it to alkaline heat treatment to form a slurry; sequentially adding Bacillus licheniformis alkaline protease, trypsin, and pepsin to the slurry for three separate hydrolysis processes to obtain a hydrolysate; after enzyme inactivation treatment of the hydrolysate, adding Lactobacillus plantarum for fermentation to obtain a fermentation broth; sterilizing the fermentation broth and then filtering it to obtain a fermentation supernatant; sequentially subjecting the fermentation supernatant to membrane filtration, sterilization, and spray drying to obtain the active polypeptide of chicken gizzard lining. The active polypeptide of chicken gizzard lining prepared by the method of this invention exhibits alcohol dehydrogenase activating activity, AGEs inhibitory activity, albumin denaturation inhibitory activity, lipoxygenase inhibitory activity, pancreatic lipase inhibitory activity, and a beneficial bacteria proliferation promoting effect.

Description

Technical Field

[0001] This invention relates to the field of chicken gizzard processing technology, and in particular to an active polypeptide of chicken gizzard and its preparation method and functional products. Background Technology

[0002] Chicken gizzard lining, recorded in the "Shennong's Classic of Materia Medica" as a superior medicine from the neck of a rooster, contains protein, polysaccharides, amino acids, trace elements, etc. Its main functions are to strengthen the stomach and aid digestion, astringe essence and stop seminal emission, promote urination and dissolve stones, and prevent arteriosclerosis. It is widely used in internal medicine, surgery, gynecology, and pediatrics.

[0003] Chicken gizzard lining, as a food and medicinal ingredient rich in high-quality protein, currently lacks comprehensive utilization through deep processing to prepare bioactive peptides, and related research is scarce. For example:

[0004] Zhang Huiying et al. used Bacillus subtilis neutral protease and Bacillus licheniformis alkaline protease to enzymatically hydrolyze chicken gizzard lining, studied the optimal enzymatic hydrolysis process conditions for chicken gizzard lining, and analyzed the amino acid composition, molecular weight distribution and antioxidant activity of the hydrolysate. However, the enzymatic hydrolysis effect was not good and the yield was low.

[0005] Chinese invention patent application CN110272934 A, entitled "Extraction Method and Application of Small Molecule Peptides from Chicken Gizzard Membrane," describes the extraction of small molecule peptides from chicken gizzard membrane via enzymatic hydrolysis using trypsin and a complex flavor protease. However, the physiological functions and biological activities of these small molecule peptides are unknown; only that the extracted peptides have high protein content is known.

[0006] Chinese invention patent application CN113616678A, entitled "A Chicken Gizzard Enzymatic Hydrolysate and Its Preparation Method and Use", describes the preparation of a chicken gizzard enzymatic hydrolysate using enzymatic hydrolysis technology. The prepared extract contains nine essential amino acids, low-molecular-weight polysaccharides, and polypeptides, among other bioactive small molecules. However, the specific physiological functions and bioactivity of the hydrolysate are unknown. It is only known that the prepared medium contains bioactive small molecules such as amino acids, low-molecular-weight polysaccharides, and polypeptides, indicating that it has a digestive-promoting function. The physiological effects and functions of the polypeptides are still unknown.

[0007] No reports have been found regarding fermented chicken gizzard lining bioactive peptides, nor have any research reports been found on fermented chicken gizzard lining bioactive peptides exhibiting alcohol dehydrogenase activating activity, AGEs inhibitory activity, albumin denaturation inhibitory activity, lipoxygenase inhibitory activity, pancreatic lipase inhibitory activity, or lactic acid bacteria proliferation-promoting activity. Furthermore, there are no publicly available reports on extraction methods for peptides with the aforementioned bioactive functions. Therefore, there is a research gap in the current field regarding how to prepare chicken gizzard lining bioactive peptides with the aforementioned bioactive functions. Summary of the Invention

[0008] To address the gaps in the prior art mentioned above, this invention provides a method for preparing chicken gizzard membrane active polypeptides, the technical solution of which is as follows:

[0009] The preparation method of this chicken gizzard active polypeptide includes the following steps:

[0010] After crushing the chicken gizzard lining, add it to water and perform alkaline heat treatment to make a slurry;

[0011] Bacillus licheniformis alkaline protease, trypsin, and pepsin were added to the slurry in sequence for three separate hydrolysis processes to obtain a hydrolysate.

[0012] After the hydrolysate is treated to remove enzymes, Lactobacillus plantarum is added for fermentation to obtain a fermentation broth.

[0013] After sterilization, the fermentation broth is filtered to obtain the fermentation supernatant.

[0014] The fermentation supernatant was sequentially subjected to membrane filtration, sterilization, and spray drying to obtain the chicken gizzard active polypeptide.

[0015] Among them, the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum BXM2, with accession number CGMCC NO.16436.

[0016] In some embodiments, the slurry undergoes three hydrolysis processes, including the following steps: after adjusting the pH of the slurry, Bacillus licheniformis alkaline protease is added for a first hydrolysis to obtain a first hydrolysate; wherein the conditions for the first hydrolysis are: constant temperature hydrolysis for 100-150 min at a system pH of 8.0-9.0 and a temperature of 50-55°C; after adjusting the pH of the first hydrolysate, trypsin is added for a second hydrolysis to obtain a second hydrolysate; wherein the conditions for the second hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 7.0-8.0 and a temperature of 37-40°C; after adjusting the pH of the second hydrolysate, pepsin is added for a third hydrolysis to obtain a third hydrolysate; wherein the conditions for the third hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 1.0-4.0 and a temperature of 37-40°C.

[0017] In some embodiments, the amount of Bacillus licheniformis alkaline protease added is 4000-10000 U / g, based on the protein content of the chicken gizzard raw material; the amount of trypsin added is 1000-3000 U / g, based on the protein content of the chicken gizzard raw material; and the amount of pepsin added is 10-40 U / g, based on the protein content of the chicken gizzard raw material.

[0018] In some embodiments, the ratio of Lactobacillus plantarum BXM2 to the hydrolysate is (0.001-0.03) g: 1 ml, the fermentation temperature is 35-40°C, and the fermentation time is 18-24 h.

[0019] In some embodiments, chicken gizzard powder is pulverized and added to water, and the reaction system is subjected to alkaline heat treatment at pH 9-11 and temperature 75-80℃ for 1-3 hours to prepare a slurry; wherein, the ratio of chicken gizzard powder to water is 1g:(10-15)ml.

[0020] In some embodiments, after the fermentation broth is sterilized, the fermentation supernatant and bacterial sludge are separated by plate and frame filtration to obtain the fermentation supernatant.

[0021] In some embodiments, the fermentation supernatant is membrane filtered to retain polypeptides with a molecular weight of less than 5000 Da; the polypeptides are sterilized at (0.1-0.2) MPa and (105-121) °C for (15-20) min, and then spray-dried.

[0022] In some embodiments, the spray drying conditions are: outlet air temperature 90-95℃, inlet air temperature 180-185℃, atomization frequency 350Hz, induced draft fan frequency 40-45Hz, and tower pressure upper and lower limits of -1250-1250Pa.

[0023] The present invention also provides a functional product, the components of which include chicken gizzard active polypeptides prepared by the preparation method described above.

[0024] In some embodiments, the functional product includes functional foods, health supplements, cosmetics, and skincare products; the functional product includes at least one of the following functions:

[0025] (1) It has alcohol dehydrogenase activating activity;

[0026] (2) AGEs inhibit activity;

[0027] (3) It has albumin denaturation inhibitory activity and lipoxygenase inhibitory activity;

[0028] (4) It has pancreatic lipase inhibitory activity;

[0029] (5) It has beneficial bacteria promoting activity.

[0030] Based on the above, compared with the prior art, the method for preparing chicken gizzard membrane active polypeptide of the present invention has the following beneficial effects:

[0031] This invention involves the extraction and purification of peptides from chicken gizzard lining to obtain bioactive peptides exhibiting activity activating alcohol dehydrogenase, inhibiting AGEs, inhibiting albumin denaturation, inhibiting lipoxygenase, inhibiting pancreatic lipase, and promoting the proliferation of beneficial bacteria. These bioactive peptides can be used as functional factors in products with effects such as hangover relief, liver protection, anti-inflammation, weight loss, anti-glycation, and regulating intestinal health. This invention provides a theoretical basis and practical solution for the deep processing and industrialization of chicken gizzard lining.

[0032] This active polypeptide from chicken gizzard lining is prepared by a combination of enzymatic hydrolysis with specific enzymes and fermentation with specific bacterial strains. When applied to food, this active polypeptide has a good taste and flavor, which helps to improve the user experience.

[0033] The method of this invention can produce the desired polypeptide by using a simple combination of operations such as enzymatic hydrolysis, fermentation, and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0034] 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 particularly pointed out in the description and claims. Detailed Implementation

[0035] 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 in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. 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.

[0036] 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.

[0037] This invention provides an operational example of a method for preparing active polypeptides from chicken gizzard membrane, the specific steps of which are as follows:

[0038] Step 1.

[0039] After crushing the chicken gizzard lining, add it to water and perform alkaline heat treatment to make a slurry;

[0040] The process involves pulverizing chicken gizzard lining and adding it to water. The reaction system is then subjected to alkaline heat treatment at pH 9–11 and temperature 75–80℃ for 1–3 hours to prepare a slurry. The ratio of chicken gizzard lining powder to water is 1g:(10–15)ml.

[0041] Step 2.

[0042] Bacillus licheniformis alkaline protease, trypsin, and pepsin were added to the slurry in sequence for three separate hydrolysis processes to obtain a hydrolysate.

[0043] The slurry undergoes three hydrolysis processes, including: adjusting the pH of the slurry, adding Bacillus licheniformis alkaline protease for a first hydrolysis to obtain a first hydrolysate; wherein the conditions for the first hydrolysis are: constant temperature hydrolysis for 100-150 min at a system pH of 8.0-9.0 and a temperature of 50-55℃; adjusting the pH of the first hydrolysate, adding trypsin for a second hydrolysis to obtain a second hydrolysate; wherein the conditions for the second hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 7.0-8.0 and a temperature of 37-40℃; adjusting the pH of the second hydrolysate, adding pepsin for a third hydrolysis to obtain a third hydrolysate; wherein the conditions for the third hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 1.0-4.0 and a temperature of 37-40℃; the stirring and shearing speed for each of the three hydrolysis processes is 800-2000 r / min.

[0044] The amount of Bacillus licheniformis alkaline protease added is 4000-10000 U / g, based on the protein content of the chicken gizzard raw material; the amount of trypsin added is 1000-3000 U / g, based on the protein content of the chicken gizzard raw material; and the amount of pepsin added is 10-40 U / g, based on the protein content of the chicken gizzard raw material.

[0045] Step 3.

[0046] After the hydrolysate is treated to remove enzymes, Lactobacillus plantarum is added for fermentation to obtain a fermentation broth.

[0047] The ratio of *Lactobacillus plantarum* BXM2 to the hydrolysate was (0.001–0.03) g:1 ml, the fermentation temperature was 35–40℃, and the fermentation time was 18–24 h. The *Lactobacillus plantarum* was *Lactobacillus plantarum* (…). Lactobacillus plants BXM2, with accession number CGMCC NO.16436.

[0048] Step 4.

[0049] After sterilization, the fermentation broth is separated into fermentation supernatant and bacterial sludge by plate and frame filtration to obtain fermentation supernatant.

[0050] Step 5.

[0051] The fermentation supernatant was membrane filtered to retain polypeptides with a molecular weight of less than 5000 Da; the polypeptides were sterilized at (0.1-0.2) MPa and (105-121) °C for (15-20) min, and then spray-dried to obtain the chicken gizzard active polypeptide.

[0052] The spray drying conditions are as follows: outlet air temperature 90-95℃, inlet air temperature 180-185℃, atomization frequency 350Hz, induced draft fan frequency 40-45Hz, and tower pressure upper and lower limits of -1250-1250Pa.

[0053] 6. Activity test: The active peptides of the inner metal were tested.

[0054] Chicken gizzard peptides obtained by enzymatic fermentation technology have alcohol dehydrogenase activating activity, AGEs inhibitory activity, albumin denaturation inhibitory activity, lipoxygenase inhibitory activity, pancreatic lipase inhibitory activity, and beneficial bacteria promoting activity.

[0055] The present invention also provides the following embodiments and comparative examples to verify the effectiveness of the present invention:

[0056] The enzyme activities of *Bacillus licheniformis* alkaline protease (400,000 U / g), *Bacillus subtilis* neutral protease (80,000 U / g), trypsin (250,000 U / g), pepsin (3,000 U / g), papain (800,000 U / g), and aminopeptidase (5,000 U / g) used in the examples and comparative examples were as follows: The unit "U / g" refers to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a specific chemical reaction), which differs from the meaning of the unit "U / g" used to calculate the enzyme addition amount based on the protein content per gram of chicken gizzard powder. Here, XU / g enzyme activity means: XU of enzyme activity per gram of enzyme.

[0057] The enzyme addition amount mentioned in this article, calculated based on the raw material protein, is expressed in units of XU / g, which means: for 1g of raw material protein, the corresponding enzyme activity to be added is XU.

[0058] Example 1

[0059] 1. Raw material pretreatment: 100g of chicken gizzard lining (85% protein content) was pulverized and added to purified water at a mass-to-volume ratio of 1:15 (g:ml). The pH of the solution was adjusted to 11, and then subjected to alkaline heat treatment at 80℃ for 2 hours with a stirring and shearing speed of 1000 r / min to prepare a slurry. Here, the initial pH of the system is 11. The pH will change during the subsequent alkaline heat reaction; therefore, alkali needs to be added to adjust the pH to 9 during the first hydrolysis.

[0060] 2. First hydrolysis: The pH of the chicken gizzard solution system was adjusted to 9.0 using sodium hydroxide. Based on the protein content of the raw materials, 9412 U / g (i.e. 2g) of Bacillus licheniformis alkaline protease was added. The solution was hydrolyzed at a constant temperature of 55℃ for 120 min with a stirring and shearing speed of 1000 r / min to obtain the first hydrolysate.

[0061] 3. Secondary hydrolysis: Adjust the system temperature to 37℃ and the pH value to 8.0. Add 1471U / g of trypsin according to the protein content of the raw material. Hydrolyze at a constant temperature for 60 minutes and stir at a shear rate of 1000r / min to obtain the second hydrolysate.

[0062] 4. Third hydrolysis: Keeping the temperature constant, adjust the pH of the system to 4.0, add 17.65 U / g of pepsin based on the protein content of the raw material, and hydrolyze at a constant temperature for 60 min with a stirring shear rate of 1000 r / min to obtain the third hydrolysate. After enzymatic hydrolysis, heat to above 90℃ for 15 minutes to inactivate the enzyme.

[0063] 5. Microbial fermentation: Control the fermentation temperature at 37℃, add 3.0% (g:ml) of Lactobacillus plantarum BXM2 by mass of hydrolysate, and ferment in a constant temperature incubator for 20h.

[0064] 6. Filtration and purification: After fermentation, sterilization was performed (121℃ for 20 min). Then, the fermentation supernatant and bacterial sludge were separated by plate and frame filtration. The fermentation broth was treated with a 5000Da ultrafiltration membrane to obtain chicken gizzard active peptides with a molecular weight of less than 5000Da.

[0065] 7. Spray drying: The peptides are sterilized at 121℃ for 20 minutes under 0.1 MPa, and then spray dried. The outlet air temperature of the spray drying device is controlled at 90℃, the inlet air temperature at 180℃, the atomization frequency at 350 Hz, the induced draft fan frequency at 40-45 Hz, and the upper and lower limits of the tower pressure at -1250-1250 Pa, to obtain chicken gizzard active peptide powder.

[0066] Example 2

[0067] 1. Raw material pretreatment: 100g of chicken gizzard lining (protein content 85%) was crushed and added to purified water at a mass-volume ratio of 1:12 (g:ml). The pH of the solution was adjusted to 11, and then the mixture was subjected to alkaline heat treatment at 80℃ for 3 hours with a stirring and shearing speed of 1000r / min to prepare a slurry. Here, the initial pH of the system is 11. The acid-base value will change during the subsequent alkaline heat reaction. Therefore, alkali needs to be added to adjust the pH of the system to 8 during the first hydrolysis.

[0068] 2. Single hydrolysis: Adjust the pH of the chicken gizzard lining solution to 8.0 using sodium hydroxide. Add 4706 U / g of Bacillus licheniformis alkaline protease based on the protein content of the raw materials. Hydrolyze at a constant temperature of 55℃ for 120 min with a stirring shear rate of 800 r / min.

[0069] 3. Secondary hydrolysis: Adjust the system temperature to 37℃ and pH to 8.0. Add 2942 U / g of trypsin based on the protein content of the raw materials. Hydrolyze at a constant temperature for 60 min and stir at a shear rate of 800 r / min.

[0070] 4. Three-stage hydrolysis: Maintain a constant temperature of 37℃, adjust the pH of the system to 4.0, add 35.30 U / g of pepsin based on the protein content of the raw material, and hydrolyze at a constant temperature for 60 minutes with a stirring shear rate of 1000 r / min. After enzymatic hydrolysis, heat to above 90℃ for 15 minutes to inactivate the enzyme.

[0071] 5. Microbial fermentation: Control the fermentation temperature at 37℃, add 2.0% (g:ml) of Lactobacillus plantarum BXM2 by mass of hydrolysate, and ferment in a constant temperature incubator for 24 hours.

[0072] 6. Filtration and purification: After fermentation, sterilization was performed (121℃ for 20 min). Then, the fermentation supernatant and bacterial sludge were separated by plate and frame filtration. The fermentation broth was treated with a 5000Da ultrafiltration membrane to obtain chicken gizzard active peptides with a molecular weight of less than 5000Da.

[0073] 7. Spray drying: The peptides are sterilized at 121℃ for 20 minutes under 0.1 MPa, and then spray dried. The outlet air temperature of the spray drying device is controlled at 90℃, the inlet air temperature at 180℃, the atomization frequency at 350 Hz, the induced draft fan frequency at 40-45 Hz, and the upper and lower limits of the tower pressure at -1250-1250 Pa, to obtain chicken gizzard active peptide powder.

[0074] Example 3

[0075] 1. Raw material pretreatment: 100g of chicken gizzard lining (protein content 85%) was crushed and added to purified water at a mass-volume ratio of 1:8 (g:ml). The pH of the solution was adjusted to 9, and then the mixture was subjected to alkaline heat treatment at 80℃ for 2 hours with a stirring and shearing speed of 800r / min to prepare a slurry. Here, the initial pH of the system is 9. The acid-base value will change during the subsequent alkaline heat reaction. Therefore, alkali needs to be added to adjust the pH of the system to 9 during the first hydrolysis.

[0076] 2. Single hydrolysis: Adjust the pH of the chicken gizzard lining solution to 9.0 using sodium hydroxide. Add 7059 U / g of Bacillus licheniformis alkaline protease based on the protein content of the raw materials. Hydrolyze at a constant temperature of 55℃ for 120 min with a stirring and shearing speed of 1000 r / min.

[0077] 3. Secondary hydrolysis: Adjust the system temperature to 37℃ and pH to 7.0. Add 1471U / g of trypsin based on the protein content of the raw materials. Hydrolyze at a constant temperature for 60 minutes and stir at a shear rate of 800r / min.

[0078] 4. Three-stage hydrolysis: Keep the temperature constant, adjust the pH of the system to 4.0, add 17.65 U / g of pepsin based on the protein content of the raw material, and hydrolyze at a constant temperature for 60 minutes with a stirring shear rate of 1000 r / min. After enzymatic hydrolysis, heat to above 90℃ for 15 minutes to inactivate the enzyme.

[0079] 5. Microbial fermentation: Control the fermentation temperature at 40℃, add 0.5% (g:ml) of Lactobacillus plantarum BXM2 by mass of hydrolysate volume, and ferment in a constant temperature incubator for 22h.

[0080] 6. Filtration and purification: After fermentation, sterilization was performed (121℃ for 20 min). Then, the fermentation supernatant and bacterial sludge were separated by plate and frame filtration. The fermentation broth was treated with a 5000Da ultrafiltration membrane to obtain chicken gizzard active peptides with a molecular weight of less than 5000Da.

[0081] 7. Spray drying: The peptides are sterilized at 121℃ for 20 minutes under 0.1 MPa, and then spray dried. The outlet air temperature of the spray drying device is controlled at 90℃, the inlet air temperature at 180℃, the atomization frequency at 350 Hz, the induced draft fan frequency at 40-45 Hz, and the upper and lower limits of the tower pressure at -1250-1250 Pa, to obtain chicken gizzard active peptide powder.

[0082] Example 4

[0083] 1. Raw material pretreatment: 100g of chicken gizzard lining (protein content 85%) was crushed and added to purified water at a mass-volume ratio of 1:10 (g:ml). The pH of the solution was adjusted to 11, and then the mixture was subjected to alkaline heat treatment at 80℃ for 2 hours with a stirring and shearing speed of 1000r / min to prepare a slurry. Here, the initial pH of the system is 11. The acid-base value will change during the subsequent alkaline heat reaction. Therefore, alkali needs to be added to adjust the pH of the system to 9 during the first hydrolysis.

[0084] 2. Single hydrolysis: Adjust the pH of the chicken gizzard lining solution to 9.0 using sodium hydroxide. Add 7059 U / g of Bacillus licheniformis alkaline protease based on the protein content of the raw materials. Hydrolyze at a constant temperature of 55℃ for 120 min with a stirring and shearing speed of 1000 r / min.

[0085] 3. Secondary hydrolysis: Adjust the system temperature to 37℃ and pH to 8.0. Add 1471U / g of trypsin based on the protein content of the raw materials. Hydrolyze at a constant temperature for 60 minutes and stir at a shear rate of 800r / min.

[0086] 4. Three-stage hydrolysis: Keep the temperature constant, adjust the pH of the system to 4.0, add 35.30 U / g of pepsin based on the protein content of the raw material, and hydrolyze at a constant temperature for 90 minutes with a stirring shear rate of 800 r / min. After enzymatic hydrolysis, heat to above 90℃ for 15 minutes to inactivate the enzyme.

[0087] 5. Microbial fermentation: Control the fermentation temperature at 37℃, add 2.0% (g:ml) of Lactobacillus plantarum BXM2 by mass of hydrolysate, and ferment in a constant temperature incubator for 18h.

[0088] 6. Filtration and purification: After fermentation, sterilization was performed (121℃ for 20 min). Then, the fermentation supernatant and bacterial sludge were separated by plate and frame filtration. The fermentation broth was treated with a 5000Da ultrafiltration membrane to obtain chicken gizzard active peptides with a molecular weight of less than 5000Da.

[0089] 7. Spray drying: The peptides are sterilized at 121℃ for 20 minutes under 0.1 MPa, and then spray dried. The outlet air temperature of the spray drying device is controlled at 90℃, the inlet air temperature at 180℃, the atomization frequency at 350 Hz, the induced draft fan frequency at 40-45 Hz, and the upper and lower limits of the tower pressure at -1250-1250 Pa, to obtain chicken gizzard active peptide powder.

[0090] Example 5

[0091] 1. Raw material pretreatment: 100g of chicken gizzard lining (protein content 85%) was crushed and added to purified water at a mass-volume ratio of 1:15 (g:ml). The pH of the solution was adjusted to 11, and then the mixture was subjected to alkaline heat treatment at 80℃ for 3 hours with a stirring and shearing speed of 1000r / min to prepare a slurry. Here, the initial pH of the system is 11. The acid-base value will change during the subsequent alkaline heat reaction. Therefore, alkali needs to be added to adjust the pH of the system to 8 during the first hydrolysis.

[0092] 2. Single hydrolysis: Adjust the pH of the chicken gizzard lining solution to 8.0 using sodium hydroxide. Add 4706 U / g of Bacillus licheniformis alkaline protease based on the protein content of the raw materials. Hydrolyze at a constant temperature of 55℃ for 150 min with a stirring shear rate of 800 r / min.

[0093] 3. Secondary hydrolysis: Adjust the system temperature to 37℃ and pH to 8.0. Add 1471U / g of trypsin based on the protein content of the raw materials. Hydrolyze at a constant temperature for 60 minutes and stir at a shear rate of 800r / min.

[0094] 4. Three-stage hydrolysis: Keep the temperature constant, adjust the pH of the system to 3.0, add 17.65 U / g of pepsin based on the protein content of the raw material, and hydrolyze at a constant temperature for 60 minutes with a stirring shear rate of 800 r / min. After enzymatic hydrolysis, heat to above 90℃ for 15 minutes to inactivate the enzyme.

[0095] 5. Microbial fermentation: Control the fermentation temperature at 40℃, add 3.0% (g:ml) of Lactobacillus plantarum BXM2 by mass of hydrolysate, and ferment in a constant temperature incubator for 24 hours.

[0096] 6. Filtration and purification: After fermentation, sterilization was performed (121℃ for 20 min). Then, the fermentation supernatant and bacterial sludge were separated by plate and frame filtration. The fermentation broth was treated with a 5000Da ultrafiltration membrane to obtain chicken gizzard active peptides with a molecular weight of less than 5000Da.

[0097] 7. Spray drying: The peptides are sterilized at 121℃ for 20 minutes under 0.1 MPa, and then spray dried. The outlet air temperature of the spray drying device is controlled at 90℃, the inlet air temperature at 180℃, the atomization frequency at 350 Hz, the induced draft fan frequency at 40-45 Hz, and the upper and lower limits of the tower pressure at -1250-1250 Pa, to obtain chicken gizzard active peptide powder.

[0098] Comparative Example 1

[0099] Compared to Example 1, the chicken gizzard hydrolysate was not fermented, i.e., step 5 was not performed, and the remaining operations and processes were the same as in Example 1.

[0100] Comparative Example 2

[0101] Compared to Example 1, the fermentation strain of the chicken gizzard hydrolysate was replaced with Lactobacillus paracasei YYS-69 (CGMCC No. 25837) of equal quality, while all other aspects were the same as in Example 1.

[0102] Comparative Example 3

[0103] Compared to Example 1, the fermentation strain of chicken gizzard hydrolysate was replaced with Lactobacillus plantarum YYS-99 (CGMCC No. 25838) of equal quality, while all other aspects were the same as in Example 1.

[0104] Comparative Example 4

[0105] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease. The amount of Bacillus licheniformis alkaline protease used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1, and the hydrolysis conditions are the same as those of Bacillus licheniformis alkaline protease hydrolysis in Example 1. In addition, the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as in Example 1.

[0106] Specifically, the enzyme activity replacement process is as follows:

[0107] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 2g. Based on the protein content in the chicken gizzard powder, the amount of Bacillus licheniformis alkaline protease added was 9412U / g. The specific calculation process is as follows:

[0108] The enzyme activity of 2g of Bacillus licheniformis alkaline protease is 400,000 U / g * 2g = 800,000 U. Converted to the enzyme addition amount based on the protein content per g of chicken gizzard powder, it is 800,000 U / (100g * 85%)g = 9412 U / g.

[0109] In Example 1, the amount of trypsin added was 0.5g. Based on the protein content in the chicken gizzard powder, the amount of trypsin added was 1470.59U / g. The specific calculation process is as follows:

[0110] The enzyme activity of 0.5g of trypsin is 250,000 U / g * 0.5g = 125,000 U. Converted to the amount of enzyme added per gram of chicken gizzard powder based on the protein content, it is 125,000 U / (100g * 85%)g = 1470.59 U / g.

[0111] In Example 1, the amount of pepsin added was 0.5g. Based on the protein content in the chicken gizzard powder, the amount of pepsin added was 17.65U / g. The specific calculation process is as follows:

[0112] The enzyme activity of 0.5g of pepsin is 3000U / g * 0.5g = 1500U. Converted to the amount of enzyme added per gram of chicken gizzard powder, it is 1500U / (100g * 85%)g = 17.65U / g.

[0113] Therefore, based on the protein content of the raw materials, the amount of Bacillus licheniformis alkaline protease added in this comparative example is 10900.24 U / g, which is 2.32g.

[0114] Compared with Example 1

[0115] The total amount of alkaline protease, trypsin, and pepsin from Bacillus licheniformis, and their corresponding enzyme activities. The conversion method for the amount of other enzymes added is the same as the above calculation method.

[0116] Comparative Example 5

[0117] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard membrane only involves trypsin hydrolysis, and the amount of trypsin used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1. The hydrolysis conditions are the same as those of trypsin hydrolysis in Example 1, and the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as those of Example 1.

[0118] Based on the protein content of the raw materials, the amount of trypsin added in this comparative example is 10900.24 U / g, or 3.706g.

[0119] Comparative Example 6

[0120] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard membrane only involves pepsin hydrolysis, and the amount of pepsin used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1. The hydrolysis conditions are the same as those of pepsin hydrolysis in Example 1, and the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as those of Example 1.

[0121] Based on the protein content of the raw materials, the amount of pepsin added in this comparative example is 10900.24 U / g, or 308.85g.

[0122] Comparative Example 7

[0123] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease and trypsin. The amount of Bacillus licheniformis alkaline protease used for hydrolysis remains unchanged, while the amount of trypsin used for hydrolysis is replaced by an equal enzyme activity amount to replace the total amount of enzymes used in the second and third hydrolysis in Example 1. The hydrolysis conditions for Bacillus licheniformis alkaline protease and trypsin hydrolysis remain unchanged, and the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as in Example 1.

[0124] After conversion, the amount of Bacillus licheniformis alkaline protease used in this comparative example is 2g. Based on the protein content of the raw materials, the amount of trypsin added is 1488.65U / g, which is 0.5061g.

[0125] Comparative Example 8

[0126] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease and pepsin. The amount of Bacillus licheniformis alkaline protease used for hydrolysis remains unchanged, and trypsin hydrolysis is not performed. The amount of pepsin used for hydrolysis is the same as the total amount of enzymes used in the second and third hydrolysis in Example 1. The hydrolysis conditions for Bacillus licheniformis alkaline protease and pepsin hydrolysis remain unchanged, and the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as in Example 1.

[0127] After conversion, the amount of Bacillus licheniformis alkaline protease used in this comparative example is 2g. Based on the protein content of the raw materials, the amount of pepsin added is 1488.65U / g, which is 42.178g.

[0128] Comparative Example 9

[0129] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard membrane only involves trypsin and pepsin hydrolysis, without Bacillus licheniformis alkaline protease hydrolysis. The amount of trypsin used for trypsin hydrolysis is the same as the total amount of enzyme in the first and second hydrolysis in Example 1, while the amount of pepsin used for pepsin hydrolysis remains unchanged. The hydrolysis conditions for trypsin and pepsin hydrolysis remain unchanged, and the hydrolysate does not undergo microbial fermentation. All other operations and processes are the same as in Example 1.

[0130] After conversion, the amount of pepsin used in this comparative example is 0.5g. Based on the protein content of the raw materials, the amount of trypsin added is 10883U / g, which is 3.700g.

[0131] Comparative Example 10

[0132] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease. The amount of Bacillus licheniformis alkaline protease used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1, and the hydrolysis conditions are the same as those of Bacillus licheniformis alkaline protease hydrolysis in Example 1. All other operations and processes are the same as in Example 1.

[0133] Based on the protein content of the raw materials, the amount of Bacillus licheniformis alkaline protease added in this comparative example is 10900.24 U / g, or 2.32g.

[0134] Comparative Example 11

[0135] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard membrane only involves trypsin hydrolysis, and the amount of trypsin used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1, and the hydrolysis conditions are the same as the trypsin hydrolysis conditions of Example 1. All other operations and processes are the same as in Example 1.

[0136] Based on the protein content of the raw materials, the amount of trypsin added in this comparative example is 10900.24 U / g, or 3.706g.

[0137] Comparative Example 12

[0138] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard membrane only involves pepsin hydrolysis, and the amount of pepsin used is the same as the total amount of enzyme in the three hydrolysis steps of Example 1, with the hydrolysis conditions being the same as those of Example 1. All other operations and processes are the same as those of Example 1.

[0139] Based on the protein content of the raw materials, the amount of pepsin added in this comparative example is 10900.24 U / g, or 308.85g.

[0140] Comparative Example 13

[0141] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease and trypsin. The amount of Bacillus licheniformis alkaline protease used for hydrolysis remains unchanged, while the amount of trypsin used for hydrolysis is replaced by an equal enzyme activity amount to replace the total amount of enzymes used in the second and third hydrolysis in Example 1. The hydrolysis conditions for Bacillus licheniformis alkaline protease and trypsin hydrolysis remain unchanged, and other operations and processes are the same as in Example 1.

[0142] After conversion, the amount of Bacillus licheniformis alkaline protease used in this comparative example is 2g. Based on the protein content of the raw materials, the amount of trypsin added is 1488.65U / g, which is 0.5061g.

[0143] Comparative Example 14

[0144] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining only involves hydrolysis with Bacillus licheniformis alkaline protease and pepsin. The amount of Bacillus licheniformis alkaline protease used for hydrolysis remains unchanged. Trypsin hydrolysis is not performed. The amount of pepsin used for hydrolysis is the same as the total amount of enzyme in the second and third hydrolysis in Example 1, replacing the total amount of enzyme in the second and third hydrolysis. The hydrolysis conditions for Bacillus licheniformis alkaline protease and pepsin hydrolysis remain unchanged. All other operations and processes are the same as in Example 1.

[0145] After conversion, the amount of Bacillus licheniformis alkaline protease used in this comparative example is 2g. Based on the protein content of the raw materials, the amount of pepsin added is 1488.65U / g, which is 42.178g.

[0146] Comparative Example 15

[0147] Compared to Example 1, the enzymatic hydrolysis step of chicken gizzard lining involves trypsin and pepsin hydrolysis, but not Bacillus licheniformis alkaline protease hydrolysis. The amount of trypsin used for trypsin hydrolysis is the same as the total amount of enzyme in the first and second hydrolysis in Example 1, while the amount of pepsin used for pepsin hydrolysis remains unchanged. The hydrolysis conditions for trypsin and pepsin hydrolysis remain unchanged, and other operations and processes are the same as in Example 1.

[0148] After conversion, the amount of pepsin used in this comparative example is 0.5g. Based on the protein content of the raw materials, the amount of trypsin added is 10883U / g, which is 3.700g.

[0149] Comparative Example 16

[0150] Compared to Example 1, the pepsin in the third hydrolysis was replaced with enzymes such as Bacillus subtilis neutral protease, while other operations and processes were the same as in Example 1.

[0151] Based on the protein content of the raw materials, the amount of Bacillus subtilis neutral protease added in this comparative example is 0.0188g.

[0152] Comparative Example 17

[0153] Compared to Example 1, the trypsin in the second hydrolysis was replaced with an enzyme with the activity of aminopeptidase, and the aminopeptidase hydrolysis conditions were adjusted to 55°C and pH 7.0. All other operations and processes were the same as in Example 1.

[0154] Based on the protein content of the raw materials, the amount of aminopeptidase added in this comparative example is 25.007g.

[0155] Comparative Example 18

[0156] Compared to Example 1, papain was used to replace the trypsin in the second hydrolysis in an equal amount. The papain hydrolysis conditions were 50°C and pH 6.5. All other operations and processes were the same as in Example 1.

[0157] Based on the protein content of the raw materials, the amount of papain added in this comparative example is 0.1563g.

[0158] It should be noted that:

[0159] Because the enzymes used in the above (e.g., Comparative Examples 17-18) have different applicable pH levels, the pH and hydrolysis temperature of the solutions were also adjusted to adapt to the use of different enzymes. The limiting role of environmental parameters is to adapt to the use conditions of the enzymes, not to explore and prove the influence of environmental conditions on the effect. These comparative examples are for the purpose of specifically proving the influence of enzyme type variables on the effect.

[0160] The performance of the peptide products obtained in the above examples and comparative examples was tested:

[0161] 1. Effects of polypeptide products on hangover relief function under different processing conditions

[0162] (1) Prepare a 50 mg / mL solution (solvent is water) of the sample peptide powder of the examples and comparative examples, and perform alcohol dehydrogenase activity detection on the peptide samples of the examples and comparative examples, as detailed in Table 1.

[0163] The detection reference method is as follows: Under the catalysis of alcohol dehydrogenase (ADH), ethanol reacts reversibly with oxidized coenzyme I (NAD+) to produce acetaldehyde and reduced coenzyme I (NADH). NADH has a characteristic absorption peak at 340 nm, while NAD+ does not. Using the reverse reaction: acetaldehyde + NADH → ethanol + NAD+, a decrease in absorbance at 340 nm indicates the presence of ADH enzyme activity. ADH activity is calculated by measuring the rate of change in absorbance at 340 nm.

[0164] Table 1. Ethanol dehydrogenase activity of peptide samples prepared under different preparation conditions

[0165]

[0166] In the table, "-" indicates that it was not detected.

[0167] (2) The test data (Table 1) shows that:

[0168] Examples 1-5 all exhibited good alcohol dehydrogenase activity, with Example 1 showing the highest enzyme activity at 6.11 U / mL. This may be because the combined enzymatic hydrolysis and fermentation technology fully decomposed the active polypeptides of chicken gizzard into small molecule peptides. These substances interact with ADH and, through a certain binding mechanism, can stabilize NAD+ in the reaction system, thereby enhancing the activity of alcohol dehydrogenase.

[0169] Compared with the examples, the alcohol dehydrogenase activity of Comparative Example 1 decreased to 2.25 U / mL. The reason for this may be that the active peptides of chicken gizzard lining obtained by enzymatic hydrolysis alone did not fully expose the small molecule peptides that can react with the Trp and Tyr residues of ADA to produce a transition, which significantly affected the alcohol-relieving effect of the active peptides of chicken gizzard lining.

[0170] Compared with Example 1, the alcohol dehydrogenase activity of Comparative Examples 2-18 was reduced. The data of Comparative Examples 2-3 showed that the treatment of Lactobacillus paracasei YYS-69 and Lactobacillus plantarum YYS-99 strains in the fermentation process affected the alcohol dehydrogenase activity of chicken gizzard active peptides, but the effect was not as good as that of Lactobacillus plantarum BXM2.

[0171] Compared with the examples and Comparative Example 1, the data from Comparative Examples 4-9 showed a significant decrease in alcohol dehydrogenase activity, and alcohol dehydrogenase activity was not detected in Comparative Examples 5-6. This indicates that multi-enzyme hydrolysis is more effective than single-enzyme hydrolysis. In addition, the combination of Bacillus licheniformis alkaline protease, trypsin and pepsin helps to improve alcohol dehydrogenase activity.

[0172] Comparative Examples 10-18 show that fermentation treatment affects alcohol dehydrogenase activity. The use of other enzymatic combinations (e.g., Bacillus subtilis neutral protease, aminopeptidase, papain) can alter the alcohol dehydrogenase activity of the active peptides in chicken gizzard lining, but the activity is still lower than in the examples. In the comparative examples, the effect of different fermentation strains was greater than the effect of different enzymatic combinations on alcohol dehydrogenase activity.

[0173] 2. Effects of different processing conditions on the anti-glycation ability of peptide products

[0174] (1) The peptide powder samples from the examples and comparative examples were prepared into a 50 mg / mL solution (water as the solvent). The anti-glycation performance of the peptide samples from the examples and comparative examples was tested, and the results are shown in Table 2. The method for testing the anti-glycation performance was carried out in accordance with the "Multi-model Evaluation of the Anti-glycation Effect and Active Ingredients of Sophora japonica Water Extract". The positive control was aminoguanidine 50 mg / mL.

[0175] Table 2. Inhibition rate of AGEs (Advanced Glycosylation End Products) of peptide products under different process conditions

[0176]

[0177] (2) The test data (Table 2) shows that:

[0178] Examples 1-5 all exhibited good anti-saccharification properties, with AGEs inhibition rates all above 85%. Among them, Example 1 showed better anti-saccharification properties, with an AGEs inhibition rate of 94.74%, while Example 3 had relatively lower anti-saccharification ability, with an AGEs inhibition rate of 87.02%, which may be related to the amount of enzyme and fermentation bacteria.

[0179] Compared with the examples, the anti-glycation performance of Comparative Example 1 was significantly reduced, with an AGEs inhibition rate of 46.12%. This may be because the fermentation preparation of the strain has a significant impact on the anti-glycation performance of the chicken gizzard active peptides. The examples of the present invention improve the anti-glycation ability of the chicken gizzard active peptides under the combined action of enzymatic hydrolysis and fermentation.

[0180] Compared with Example 1, the anti-glycation performance of Comparative Examples 2-18 was significantly reduced. Among them, the data of Comparative Examples 2-3 showed that different fermentation strains had a significant impact on the anti-glycation ability of chicken gizzard active peptides. This indicates that Lactobacillus paracasei YYS-69 and Lactobacillus plantarum YYS-99 may not be the dominant strains for improving the anti-glycation ability of chicken gizzard.

[0181] Comparative Examples 4-9 show that different enzymatic hydrolysis schemes have little effect on the anti-glycation properties of chicken gizzard active peptides. Comparative Examples 10-18 show that bacterial treatment helps improve the anti-glycation activity of chicken gizzard peptides. In the comparative examples with different enzymatic hydrolysis combinations than the compound enzyme hydrolysis combination of the present application, the use of Bacillus subtilis neutral protease in different enzymatic hydrolysis combinations has a greater impact on the anti-glycation properties of chicken gizzard active peptides. Its AGEs inhibition rate is the highest in the comparative examples, at 68.74%, but its anti-glycation ability is still lower than that of the enzymatic hydrolysis combination of the present application.

[0182] In summary, the reason for this may be that after the chicken gizzard lining undergoes staged enzymatic hydrolysis by Bacillus licheniformis alkaline protease, trypsin, and pepsin, the synergistic effect of the strain's fermentation inhibits protein glycosylation induced by dicarbonyl compounds, thereby reducing the amount of AGEs generated and achieving an anti-glycation effect. In contrast, the use of Bacillus subtilis neutral protease, aminopeptidase, and papain in the comparative example led to a certain degree of non-enzymatic reaction between the chicken gizzard lining protein and reducing sugars, increasing the degree of protein cross-linking and reducing the anti-glycation ability.

[0183] 3. Effects of different processing conditions on the anti-inflammatory activity of peptide products

[0184] (1) The polypeptide powders of the examples and comparative examples were prepared into a 50 mg / mL solution with water, and their albumin denaturation inhibition rate and lipoxygenase inhibition rate were detected. The results are shown in Table 3.

[0185] The determination of the albumin denaturation inhibitory activity of the active polypeptide in chicken gizzard membrane was based on the study "In vitro anti-inflammatory and antioxidant activity of an ayurvedic formulation Trayodashang guggulu". The determination of the lipoxygenase inhibitory activity was based on the study "Chemical components of the stems and leaves of Althaea sylvestris and their in vitro inhibitory activity on α-glucosidase and 5-lipoxygenase".

[0186] Table 3. Anti-inflammatory activity of peptide products under different processing conditions

[0187]

[0188] In the table, "-" indicates that it was not detected.

[0189] (2) The test data (Table 3) shows that:

[0190] Examples 1-5 all exhibited good albumin denaturation inhibition activity and lipoxygenase inhibition activity. Among them, Example 1 showed the best anti-inflammatory effect, with albumin denaturation inhibition activity and lipoxygenase inhibition activity of 98.95% and 98.03%, respectively.

[0191] Regarding albumin denaturation inhibitory activity, the examples compared with Comparative Examples 1-18 show that the albumin denaturation inhibitory activity of Comparative Examples 1-18 is reduced.

[0192] The albumin denaturation inhibition rate of Comparative Example 1 was 55.38%, which may be because the active peptides of chicken gizzard lining obtained by enzymatic hydrolysis were not fully hydrolyzed, resulting in a smaller inhibitory effect on albumin denaturation.

[0193] Comparative Examples 2 and 3 used different strains to ferment the chicken gizzard hydrolysate. The difference in fermentation degree due to the different strains may have led to a decrease in the denaturation inhibitory activity of the prepared chicken gizzard active polypeptide albumin.

[0194] Comparative Examples 4-18 used different enzymes to replace the Bacillus licheniformis alkaline protease, pepsin, and trypsin combination in Example 1 during enzymatic hydrolysis, and carried out both fermentation and non-fermentation treatments. In comparison, their albumin denaturation inhibitory activity was reduced. This may be due to the difference in enzyme cleavage sites. The peptides were hydrolyzed by pepsin and trypsin, exposing more small molecule peptides with anti-inflammatory activity, which had a greater impact on the anti-inflammatory effect of chicken gizzard active peptides.

[0195] Regarding lipoxygenase inhibitory activity, the examples compared with Comparative Examples 1-18 showed that the lipoxygenase inhibitory activity of Comparative Examples 1-18 was reduced.

[0196] Comparative Example 1, without fermentation treatment, could not obtain a polypeptide product with high lipoxygenase inhibitory activity by hydrolysis with protease alone; its lipoxygenase inhibition rate was 33.91%.

[0197] Comparative Examples 2-3 used different strains for fermentation, and compared with Example 1, the lipoxygenase inhibitory activity of their products was not improved.

[0198] Comparative Examples 4-6 used a single-enzyme hydrolysis method, and their lipoxygenase inhibition rate was not detected. Comparative Examples 7-9 used multi-enzyme hydrolysis, and their lipoxygenase inhibition effect was higher than that of the chicken gizzard active polypeptide prepared by single-enzyme treatment. However, Comparative Examples 4-9 did not undergo fermentation treatment, and their lipoxygenase inhibition activity was reduced compared with the examples. Comparative Examples 10-18 used different enzymes to replace the enzymatic hydrolysis combination in Example 1 in equal amounts for fermentation. Compared with the examples, their enzymatic hydrolysis effect significantly affected the lipoxygenase inhibition activity.

[0199] The reason for this may be that the single enzymatic hydrolysis treatment or the fermentation treatment of different strains in the comparative example cannot better expose the small molecular weight active peptides. Compared with Example 1, the specific enzymatic hydrolysis scheme of different combinations is more conducive to hydrolyzing chicken gizzard globulin and releasing peptide activity.

[0200] 4. Effects of peptide products on pancreatic lipase inhibition rate under different processing conditions

[0201] (1) Prepare a 50 mg / mL solution of the polypeptide powders of the examples and comparative examples (solvent is water) and detect the pancreatic lipase inhibition rate. See Table 4 for specific data. The detection method is in accordance with the study on the inhibitory effect of lotus leaf flavonoids on pancreatic lipase.

[0202] Table 4. Pancreatic lipase inhibition rate of peptide samples from different processes

[0203]

[0204] In the table, "-" indicates that it was not detected.

[0205] (2) The test data (Table 4) shows that:

[0206] Examples 1-5 all showed good pancreatic lipase inhibition rates, all above 75%; Example 1 showed a relatively good pancreatic lipase inhibition rate of 84.62%.

[0207] The comparison between the examples and Comparative Examples 1-18 shows that single enzymatic hydrolysis, fermentation treatment with different strains, and enzymatic hydrolysis schemes with different proteases all have a significant impact on the inhibition rate of pancreatic lipase of chicken gizzard active peptides.

[0208] The detection rate of pancreatic lipase inhibitory activity in Comparative Example 1 (unfermented) was reduced to 32.05%. This may be because the enzymatic hydrolysis of the active peptides in chicken gizzard obtained by enzymatic hydrolysis was insufficient, and some glycoprotein polymers were still present, resulting in the failure to release the effective active peptides.

[0209] The reduced pancreatic lipase inhibitory activity in Comparative Examples 2-3 indicates that fermentation with Lactobacillus paracasei YYS-69 and Lactobacillus plantarum YYS-99 after enzymatic hydrolysis cannot fully leverage the advantages of the combined enzymatic hydrolysis and fermentation technology, and cannot obtain peptides with high pancreatic lipase inhibitory activity.

[0210] Comparative Examples 4-18 were subjected to fermentation and non-fermentation treatments with different enzyme replacements. The resulting chicken gizzard active peptides exhibited certain pancreatic lipase inhibitory activity, but it was still significantly lower than that of the examples. This may be because after the alkaline protease of Bacillus licheniformis hydrolyzes some of the dextran chains in the glycoprotein, the glycoprotein structure expands, and trypsin and pepsin can better hydrolyze specific regions. After fermentation, more peptides with pancreatic lipase inhibitory activity are generated, which shows that there is a synergistic relationship between the enzymes.

[0211] 5. Effects of polypeptide products on beneficial bacteria activity under different processing conditions

[0212] (1) The active peptides of chicken gizzard membrane were used to detect the growth rate of fermented Lactobacillus mucin. The specific results are shown in Table 5. The detection method was based on the study of the in vitro growth-promoting effect of Chinese yam peptides on probiotics, with slight modifications.

[0213] The specific detection method was as follows: MRS liquid culture medium was prepared and dispensed into 10 mL tubes. 300 μL of probiotic strain (Lactobacillus fermentum) was inoculated into each tube. The experimental group was treated with 500 μL of a 20 mg / mL aqueous solution of chicken gizzard membrane active peptides, while the blank group was treated with 500 μL of sterile water. The cultures were incubated at 37°C for 24 h. The culture medium was then diluted 20-fold and the OD600 was measured to calculate the probiotic promotion rate.

[0214] Probiotic promotion rate % = (BA) / A x 100%;

[0215] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0216] Table 5. Probiotic promotion rate of peptide samples processed by different methods

[0217]

[0218] Note: - indicates not detected, meaning no effect on promoting the proliferation of beneficial bacteria. In other words, compared to the control group, it did not promote the proliferation of beneficial bacteria; the promotion rate was less than or equal to 0. The fermented *Lactobacillus fermentum* B153 was used.

[0219] (2) As can be seen from the data in Table 5:

[0220] The examples demonstrated a good effect in promoting the growth of beneficial bacteria, with a proliferation promotion rate of over 10% for fermented Lactobacillus mucinus.

[0221] Comparative Examples 1-9 did not show any promoting activity against beneficial bacteria, and Comparative Examples 10-18, although showing promoting activity, were significantly lower than those of the comparative examples. This indicates that the active polypeptide of chicken gizzard prepared by the combined fermentation of Bacillus licheniformis alkaline protease, trypsin, and pepsin using the method of the present invention has the effect of promoting the growth of beneficial bacteria.

[0222] 6. Sensory characteristics of active peptides in chicken gizzard membrane

[0223] (1) The sample peptide powders of the examples and comparative examples were prepared into solutions of 20 mg / ml (solvent was water), and their sensory characteristics were evaluated from aspects such as taste and odor. The sensory evaluation results are shown in Table 6:

[0224] Table 6 Sensory Analysis and Evaluation Table

[0225]

[0226] The sensory levels in the table are divided into "heavy, medium, light, slight, and none".

[0227] (2) From the data in Table 6, we can see that:

[0228] All examples demonstrate a good taste. The chicken gizzard active polypeptides prepared by specific enzymatic hydrolysis and fermentation with specific strains in the embodiments of the present invention have a good taste and flavor when applied to food, which is conducive to improving the user experience.

[0229] In summary, the method for preparing chicken gizzard membrane active polypeptides provided by the present invention has at least the following mechanisms of action and technical effects:

[0230] The method of this invention uses Bacillus licheniformis alkaline protease, trypsin, and pepsin for enzymatic hydrolysis, combined with Lactobacillus plantarum BXM2 fermentation to prepare chicken gizzard membrane active polypeptides. This scheme has the following effects:

[0231] (1) This invention is the first to discover that chicken gizzard active polypeptides prepared by a combination of specific enzymatic hydrolysis and specific microbial fermentation have high alcohol dehydrogenase activity, AGEs inhibition activity, albumin denaturation inhibition activity, lipoxygenase inhibition activity, pancreatic lipase inhibition activity and the effect of promoting the proliferation of beneficial bacteria. It can be used as a functional factor in functional foods, cosmetics and health products.

[0232] Among them, the active polypeptides of chicken gizzard lining have a good alcohol dehydrogenase activation rate, giving them a strong hangover-relieving effect. Alcohol dehydrogenase (ADH) plays a key role in alcohol metabolism. By activating alcohol dehydrogenase activity, the body's absorption of ethanol can be promoted, thereby relieving hangovers and protecting the liver. Under normal circumstances, more than 90% of the ethanol entering the body is metabolized by the dehydrogenase system—alcohol dehydrogenase and acetaldehyde dehydrogenase. Therefore, the active polypeptides of chicken gizzard lining have a good sobering and hangover-relieving effect and can be used in functional products (such as food and health products) with sobering, hangover-relieving, and liver-protecting effects.

[0233] As the body's sugar intake accumulates and metabolism gradually slows down, the ingested sugar easily accumulates, combines with proteins, oxidizes, and eventually forms AGEs (Advanced Glycation End Products). Chicken gizzard membrane active peptides have a strong ability to inhibit AGEs and can play an anti-glycation role, making them suitable for use in anti-glycation functional products.

[0234] Inflammation is a basic pathological process, primarily a defensive response, that occurs in living tissues with vascular systems in response to damage from various inflammatory factors. It is a natural response of the immune system to injury, infection, or other types of bodily damage and is crucial for maintaining bodily health and normal function. Based on the high albumin denaturation inhibitory activity and lipoxygenase inhibitory activity of this active peptide, it can play an anti-inflammatory role when applied to functional products (such as food and health products).

[0235] Because it inhibits the activity of lipase secreted in the intestine, it can prevent the decomposition of lipids in the intestine. Based on the high pancreatic lipase inhibitory activity of this active peptide, it can be applied to functional products (such as food and health products) to achieve functions such as weight loss.

[0236] The proliferation of fermented Lactobacillus mucin is beneficial for stomach health, digestion, and bowel movements. Based on the role of active polypeptides in chicken gizzard membrane in promoting the growth of fermented Lactobacillus mucin, its application in functional products (such as food) can play a role in aiding digestion and eliminating food stagnation.

[0237] (2) The active polypeptide of chicken gizzard is prepared by staged enzymatic hydrolysis combined with fermentation. It has no obvious bitter, fishy, ​​sour and off-odor. When it is applied to food, it has a good taste and flavor, which is conducive to improving the user experience.

[0238] (4) The method of the present invention uses a gentle biological enzyme gradient to digest protein sites, and combines simple operations such as separation and purification, molecular weight cutoff filtration, and bacterial fermentation to obtain the desired polypeptide. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0239] It should be noted that:

[0240] (1) Definition:

[0241] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0242] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. The polypeptides of this application can be used to prepare solid dosage forms such as powders, tablets, and gels, and can also be dispersed in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0243] In this article, "the effect of relieving hangovers" refers to the fact that the active polypeptides in chicken gizzard membrane can promote the absorption of ethanol by the human body by activating ADH activity, thereby relieving hangovers and protecting the liver.

[0244] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0245] The term "ultrafiltration membrane filtration" used in this article is a commonly used name for a processing step in the field, and its name accurately describes the process, so it will not be repeated here.

[0246] (2) Raw materials used in implementation:

[0247] The alkaline protease, trypsin, pepsin, papain, aminopeptidase, and neutral protease from Bacillus subtilis used are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0248] Among them, *Lactobacillus plantarum* BXM2, *Lactobacillus paracasei* YYS-69, *Lactobacillus plantarum* YYS-99, and *Lactobacillus fermentum* B153 are disclosed in Chinese invention patent applications with publication numbers CN110257306A, CN115851520A, CN115960767A, and CN113717900A, respectively. Those skilled in the art can obtain these from the depositary center based on their accession numbers. Their accession information is as follows:

[0249] Lactobacillus plantarum BXM2, Latin scientific name: Lactobacillus plantarum It is deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: September 6, 2018, accession number: CGMCC No. 16436.

[0250] Lactobacillus paracasei YYS-69, this strain is classified as Lactobacillus paracasei YYS-69, Latin scientific name: Lactobacillus Paracasei It is deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: September 28, 2022, accession number: CGMCCNo. 25837;

[0251] Lactobacillus plantarum YYS-99, this strain is classified as Lactobacillus plantarum YYS-99, Latin scientific name: Lactiplantibacillus plantarum It is deposited at the China General Microbiological Culture Collection Center, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, date of deposit: September 28, 2022, accession number: CGMCC No. 25838;

[0252] Fermented Lactobacillus mucinus uses Lactobacillus fermentum ( Lactobacillus fermentum B153 was deposited on September 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 16454, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0253] (3) Application of active polypeptides in chicken gizzard lining:

[0254] Chicken gizzard active polypeptide possesses the following characteristics: (1) it has alcohol dehydrogenase activating activity; (2) AGEs inhibitory activity; (3) it has albumin denaturation inhibitory activity and lipoxygenase inhibitory activity; (4) it has pancreatic lipase inhibitory activity; (5) it has beneficial bacteria promoting activity. Based on the above characteristics and effects, according to the functional characteristics of 1)-5), chicken gizzard active polypeptide can be applied to functional products with obvious effects such as hangover relief and liver protection, anti-glycation, anti-inflammatory, weight loss, and promoting intestinal digestion and defecation (functional products can be food, health products, cosmetics, skin care products, bath products, cleaning products, etc., including any substance that provides preventive and / or other beneficial effects). Based on the above concept, based on the correlation between the functional characteristics of chicken gizzard active polypeptide of 1)-5) and the physiological mechanisms of human or animal bodies, and the correlation with the occurrence and development of human diseases, chicken gizzard active polypeptide can also be applied to other functional products with obvious effects, including but not limited to functional products with obvious effects such as hangover relief and liver protection, anti-glycation, anti-inflammatory, weight loss, and promoting intestinal digestion and defecation.

[0255] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

[0256] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0257] 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 therein. Such 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 active polypeptide from chicken gizzard lining, characterized in that, Includes the following steps: After crushing the chicken gizzard lining, add it to water. The reaction system is then subjected to alkaline heat treatment at pH 9-11 and temperature 75-80℃ for 1-3 hours to prepare a slurry. The ratio of chicken gizzard lining powder to water is 1g:(10-15)ml. Bacillus licheniformis alkaline protease, trypsin, and pepsin were added to the slurry in sequence for three separate hydrolysis processes to obtain a hydrolysate. After the hydrolysate is treated to remove enzymes, it is fermented with *Lactobacillus plantarum* to obtain a fermentation broth; wherein, the *Lactobacillus plantarum* is *Lactobacillus plantarum* (… Lactobacillus plantarum Lactobacillus plantarum BXM2, whose preservation number is CGMCC NO.16436; in the fermentation treatment, the ratio of Lactobacillus plantarum BXM2 to the hydrolysate is (0.001~0.03) g:1 ml, the fermentation temperature is 35~40℃, and the fermentation time is 18~24 h; After sterilization, the fermentation broth is filtered to obtain the fermentation supernatant. The fermentation supernatant was filtered through a membrane to retain polypeptides with a molecular weight of less than 5000 Da; the polypeptides were sterilized at 0.1-0.2 MPa and 105-121°C for 15-20 min, and then spray-dried to obtain the chicken gizzard active polypeptide. The slurry undergoes three hydrolysis processes, including the following: After adjusting the pH of the slurry, Bacillus licheniformis alkaline protease is added for the first hydrolysis to obtain the first hydrolysate; wherein, the conditions for the first hydrolysis are: constant temperature hydrolysis for 100-150 min at a system pH of 8.0-9.0 and a temperature of 50-55℃. After adjusting the pH of the first hydrolysate, trypsin is added for a second hydrolysis to obtain a second hydrolysate; wherein the conditions for the second hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 7.0-8.0 and a temperature of 37-40℃. After adjusting the pH of the second hydrolysate, pepsin is added for a third hydrolysis to obtain a third hydrolysate; wherein the conditions for the third hydrolysis are: constant temperature hydrolysis for 60-90 min at a system pH of 1.0-4.0 and a temperature of 37-40℃. The amount of Bacillus licheniformis alkaline protease added is 4000-10000 U / g, based on the protein content of the chicken gizzard raw material; the amount of trypsin added is 1000-3000 U / g, based on the protein content of the chicken gizzard raw material; and the amount of pepsin added is 10-40 U / g, based on the protein content of the chicken gizzard raw material.

2. The method for preparing the active polypeptide of chicken gizzard membrane according to claim 1, characterized in that: After sterilization, the fermentation broth is separated into fermentation supernatant and bacterial sludge by plate and frame filtration to obtain fermentation supernatant.

3. The method for preparing the active polypeptide of chicken gizzard membrane according to claim 1, characterized in that: The spray drying conditions are as follows: outlet air temperature 90-95℃, inlet air temperature 180-185℃, atomization frequency 350Hz, induced draft fan frequency 40-45Hz, and tower pressure upper and lower limits of -1250-1250Pa.

4. A functional product, characterized in that: Its components include the active polypeptide of chicken gizzard obtained by the preparation method according to any one of claims 1-3.

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