An elastin peptide with weight loss, anti-glycation, and anti-inflammatory activities, its preparation method, and functional products thereof.

Abstract

This invention relates to the technical field of deep processing of bovine heart tubes, and particularly to an elastin peptide with weight-loss, anti-glycation, and anti-inflammatory activities, its preparation method, and functional products. The preparation method includes the following steps: mixing crushed bovine heart tubes with water and homogenizing; adding *Lactobacillus paracasei* to the homogenized liquid for fermentation; removing the accumulated oil on the surface of the fermentation liquid to obtain a defatted fermentation liquid; adding alkaline protease and trypsin sequentially to the defatted fermentation liquid for two hydrolysis processes, followed by enzyme inactivation to obtain a hydrolysate; adding activated carbon to the hydrolysate and performing solid-liquid separation to obtain a filtrate containing elastin peptides; filtering the filtrate to retain the elastin peptides, thus obtaining the elastin peptides. Using this method, elastin peptides with high glycosaminoglycan content can be obtained. These elastin peptides exhibit α-amylase inhibitory activity, antioxidant properties, pancreatic lipase inhibitory activity, anti-glycation activity, and anti-inflammatory activity, and also have a good taste and flavor.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of bovine heart tubes, and in particular to an elastin peptide with weight loss, anti-glycation and anti-inflammatory activities, its preparation method and functional products. Background Technology

[0002] Elastin is the main component of elastic fibers and is found in various connective tissues. Its amino acid sequence consists of alternating hydrophilic and hydrophobic segments, among which desmokinin and isodesmokinin are unique cross-linking structures of elastin. In human skin tissue, elastin gives the skin its elasticity and plays a stabilizing role.

[0003] Existing sources of elastin include porcine heart tubes, porcine cervical ligaments, bovine heart tubes, bovine cartilage, fish cartilage, and skipjack tuna heart bulbs. Because bovine heart tubes are widely available, they are commonly used for elastin preparation. However, bovine heart tubes contain a significant amount of fat and fibrous tissue. Currently, the process for preparing elastin from bovine heart tubes often involves removing the fat using alkali solutions. For example, Chinese invention patent application CN106381323A, published on February 8, 2017, discloses an elastin peptide and its preparation method. It discloses obtaining bovine cervical ligament elastin peptides that significantly increase the content of collagen and elastin in the skin through enzymatic hydrolysis following alkali heat treatment. However, alkali heat treatment is not very effective at removing fat in actual production and can also cause unpleasant flavor.

[0004] Furthermore, existing research on elastin peptides extracted from bovine heart tubes focuses on obtaining elastin peptides with antioxidant and skin-supporting activities, while other active functions of elastin are rarely studied. There are no reports in existing literature or patents on the preparation of elastin peptides with antioxidant, α-amylase inhibitory, pancreatic lipase inhibitory, anti-glycation, and anti-inflammatory activities using bovine heart tubes. Summary of the Invention

[0005] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing elastin peptides, the technical solution of which is as follows:

[0006] The preparation method of this elastin peptide includes the following steps:

[0007] The crushed bovine heart tubes were mixed with water and homogenized to obtain a homogenized solution.

[0008] The homogenized liquid was sterilized for the first time, and then Lactobacillus paracasei was added for fermentation to obtain a fermentation broth.

[0009] Remove the grease that has accumulated on the surface of the fermentation broth and perform a second sterilization treatment to obtain a defatted fermentation broth;

[0010] In the defatted fermentation broth, alkaline protease and trypsin are added sequentially for two hydrolysis processes, followed by enzyme inactivation treatment to obtain the hydrolysate.

[0011] The hydrolysate was subjected to solid-liquid separation to obtain a filtrate containing elastin peptides;

[0012] The filtrate is filtered to retain elastin peptides, thus obtaining the elastin peptides.

[0013] In one embodiment, the crushed bovine heart tubes are pre-treated and then mixed with water for homogenization; the pre-treatment process is as follows:

[0014] A 0.3%–0.5% sodium bicarbonate solution was added to the crushed bovine heart tubes, and the mixture was kept at 50℃–70℃ for 0.5–2 hours. Solid-liquid separation was then performed to obtain the bovine heart tube solid material. The mass ratio of the crushed bovine heart tubes to the sodium bicarbonate solution was 1:(2–4).

[0015] Water is added to the bovine heart tube solid material, and the pH of the system is adjusted to 2-4 using hydrochloric acid solution. After being kept at 70℃-90℃ for 2-4 hours, solid-liquid separation is performed, and the material is dried to obtain the pretreated bovine heart tube. The mass ratio of the crushed bovine heart tube to water is 1:(2-4).

[0016] In one embodiment, during the homogenization process, the mass ratio of the pretreated bovine heart tube to the water is 1:(5-10).

[0017] In one embodiment, the Lactobacillus paracasei is Lactobacillus paracasei YYS-K1, with accession number CGMCC No. 26405;

[0018] The mass of the Lactobacillus paracasei is (2-4) of the mass of the bovine heart tube after pretreatment.

[0019] In one embodiment, during the fermentation process, the fermentation temperature is 35–39°C and the fermentation time is 18–30 h.

[0020] In one embodiment, alkaline protease is added to the defatted fermentation broth to adjust the pH of the system to 7-9, and hydrolysis is carried out at 50-55°C for 1-3 hours, followed by enzyme inactivation treatment at 85-90°C for 20-30 minutes to obtain a first hydrolysate; wherein the mass of the alkaline protease is (2-4)% of the mass of the pretreated bovine heart tube.

[0021] Add trypsin to the first hydrolysate, adjust the pH of the system to 6.5-7.5, hydrolyze at 50-55℃ for 1-3 hours, and then inactivate the enzyme at 85-90℃ for 20-30 minutes to obtain the second hydrolysate; wherein the mass of the trypsin is (0.5-1.0)% of the mass of the bovine heart tube after the pretreatment.

[0022] In one embodiment, after the hydrolysate is treated to inactivate enzymes, activated carbon is added for further treatment, followed by filtration to obtain an elastin peptide filtrate. The filtrate is then subjected to ultrafiltration to retain elastin peptides with a molecular weight of less than 5000 Da, followed by spray drying to obtain the elastin peptides.

[0023] In one embodiment, during the treatment process, the activated carbon is added at a mass of 7-8% of the pretreated bovine heart tube mass, the treatment temperature is 50-60°C, and the treatment time is 0.5-1.5 h.

[0024] The present invention also provides an elastin peptide, which is prepared by the elastin peptide preparation method described above.

[0025] The present invention also provides a functional product, the components of which include elastin peptides prepared by the method described above; the functional product includes at least one of the following functions:

[0026] (1) It possesses α-amylase inhibitory activity;

[0027] (2) It possesses antioxidant properties;

[0028] (3) It possesses pancreatic lipase inhibitory activity;

[0029] (4) It possesses anti-glycation activity;

[0030] (5) It has anti-inflammatory activity.

[0031] Based on the above, compared with the prior art, the method for preparing elastin peptides of the present invention has the following beneficial effects:

[0032] The method of this invention can be used to prepare elastin peptides with high glycosaminoglycan content. These elastin peptides have α-amylase inhibitory activity, antioxidant activity, pancreatic lipase inhibitory activity, anti-glycation activity, and anti-inflammatory activity.

[0033] The elastin peptides prepared by this invention have no obvious bitter or sour taste, and have a good taste and flavor, which helps to improve the user experience.

[0034] The method of this invention can remove oil from the product more thoroughly and conveniently for actual production operations, thereby improving preparation efficiency and effectiveness. The solution of this invention can obtain the required elastin peptides by simply combining operations such as mixing, fermentation, enzymatic hydrolysis, and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 elastin peptides, the specific steps of which are as follows:

[0038] Step 1

[0039] After pretreatment, the broken bovine heart tubes are mixed with water and homogenized to obtain a homogenized solution.

[0040] (1) The preprocessing process is as follows:

[0041] A 0.3%–0.5% sodium bicarbonate solution was added to the crushed bovine heart tubes, and the mixture was kept at 50℃–70℃ for 0.5–2 hours. Solid-liquid separation was then performed to obtain the bovine heart tube solid material. The mass ratio of the crushed bovine heart tubes to the sodium bicarbonate solution was 1:(2–4).

[0042] Water is added to the bovine heart tube solid material, and the pH of the system is adjusted to 2-4 using hydrochloric acid solution. After being kept at 70℃-90℃ for 2-4 hours, solid-liquid separation is performed, and the material is dried to obtain the pretreated bovine heart tube. The mass ratio of the crushed bovine heart tube to water is 1:(2-4).

[0043] (2) During the homogenization process, the mass ratio of the pretreated bovine heart tube to the water is 1: (5-10).

[0044] Step Two

[0045] The homogenized liquid was sterilized for the first time, and then Lactobacillus paracasei was added for fermentation to obtain a fermentation broth. During the fermentation process, the fermentation temperature was 35-39℃ and the fermentation time was 18-30h.

[0046] The first sterilization process involves sterilizing at 85–95°C for 20–30 minutes.

[0047] Lactobacillus paracasei was Lactobacillus paracasei YYS-K1, which was deposited on January 6, 2023, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.26405; the mass of the Lactobacillus paracasei was (2-4)% of the mass of the bovine heart tube after pretreatment.

[0048] Step 3

[0049] Remove the oil accumulated on the surface of the fermentation broth and perform a second sterilization treatment to obtain a defatted fermentation broth; wherein, the second sterilization treatment process is: sterilization at 85-95℃ for 20-30 minutes.

[0050] Step Four

[0051] In the defatted fermentation broth, alkaline protease and trypsin are added sequentially for two hydrolysis processes, followed by enzyme inactivation treatment to obtain the hydrolysate; specifically:

[0052] Alkaline protease was added to the defatted fermentation broth to adjust the pH of the system to 7-9. The mixture was hydrolyzed at 50-55°C for 1-3 hours, followed by enzyme inactivation treatment at 85-90°C for 20-30 minutes to obtain the first hydrolysate. The mass of the alkaline protease was (2-4)% of the mass of the pretreated bovine heart tube.

[0053] Add trypsin to the first hydrolysate, adjust the pH of the system to 6.5-7.5, hydrolyze at 50-55℃ for 1-3 hours, and then inactivate the enzyme at 85-90℃ for 20-30 minutes to obtain the second hydrolysate; wherein the mass of the trypsin is (0.5-1.0)% of the mass of the bovine heart tube after the pretreatment.

[0054] Step 5

[0055] Activated carbon is added to the hydrolysate for treatment, followed by filtration to obtain elastin peptide filtrate. During the activated carbon treatment, the mass of the activated carbon is 7-8% of the mass of the pretreated bovine heart tube, the treatment temperature is 50-60℃, and the treatment time is 0.5-1.5h.

[0056] Step Six

[0057] The filtrate is subjected to ultrafiltration to retain elastin peptides with a molecular weight of less than 5000 Da, and then spray-dried to obtain the elastin peptides.

[0058] Step Seven

[0059] Activity assays were performed on the elastin peptides, which showed α-amylase inhibitory activity, antioxidant activity, pancreatic lipase inhibitory activity, anti-glycation and anti-inflammatory activity, and high glycosaminoglycan content.

[0060] The present invention provides the following embodiments and comparative examples.

[0061] Example 1

[0062] (1) Take 2 kg of bovine heart tube, wash it and mince it, add 6 kg of 0.3% sodium bicarbonate solution, keep it at 60℃ for 1 hour and then wash it with pure water;

[0063] (2) Add 6 kg of water to the cleaned beef heart tube mince, adjust the pH to 3 with hydrochloric acid, keep it at 85℃ for 2 hours, wash and dry it for later use.

[0064] (3) Take 1 kg of pretreated dried bovine heart tube fragments (80% protein content), add 5 kg of water for homogenization to obtain bovine heart tube homogenate, sterilize at 95℃ for 30 min, add 30 g of Lactobacillus paracasei YYS-K1 (CGMCC No.26405) and ferment at 37℃ for 24 h;

[0065] (4) After fermentation, the oil accumulated on the surface of the bovine heart tube fermentation liquid was removed by filtration, and the liquid was sterilized at 95°C for 30 minutes to obtain defatted bovine heart tube fermentation liquid.

[0066] (5) After the defatted bovine heart tube fermentation broth is cooled to 55°C, add 30g of alkaline protease (the amount of alkaline protease added is 3% of the mass of the pretreated bovine heart tube) for the first enzymatic hydrolysis. The enzymatic hydrolysis pH is 8.0, the enzymatic hydrolysis time is 2h, and the enzyme is inactivated at 90°C for 30min. The alkaline protease is Bacillus licheniformis alkaline protease. The enzyme activity of alkaline protease is 200,000 U / g, which is expressed as 200,000 U of enzyme activity per gram of enzyme.

[0067] After enzyme inactivation was completed and the temperature was lowered to 55℃, 6.4g of trypsin (the amount of trypsin added was 0.64% of the mass of the pretreated bovine heart tube) was added for a second enzymatic hydrolysis. The hydrolysis pH was 7.5, the hydrolysis time was 2h, and the enzyme was inactivated at 90℃ for 20min. The enzyme activity of trypsin was 100,000 U / g, which is expressed as 100,000 U of enzyme activity per gram of enzyme.

[0068] (6) Add 75g of activated carbon, keep warm at 55℃ for 1h, then filter by plate and frame to obtain elastin peptide filtrate:

[0069] (7) The filtrate is treated with a 5000 Da ultrafiltration membrane to obtain a bovine heart tube elastin solution with a molecular weight of less than 5000 Da, and then spray-dried to obtain elastin peptides.

[0070] Example 2

[0071] (1) Take 2 kg of bovine heart tube, wash it and mince it, add 6 kg of 0.3% sodium bicarbonate solution, keep it at 70℃ for 1 hour and then wash it with pure water;

[0072] (2) Add 6 kg of water to the cleaned beef heart tube mince, adjust the pH to 4 with hydrochloric acid, keep it at 85℃ for 2 hours, wash and dry it for later use.

[0073] (3) Take 1 kg of pretreated dried bovine heart tube fragments (80% protein content), add 5 kg of water for homogenization to obtain bovine heart tube homogenate, sterilize at 95℃ for 30 min, add 20 g of Lactobacillus paracasei YYS-K1 (CGMCC No.26405) and ferment at 35℃ for 30 h;

[0074] (4) After fermentation, remove the oil accumulated on the surface of the bovine heart tube fermentation liquid, sterilize at 95°C for 30 minutes to obtain defatted bovine heart tube fermentation liquid;

[0075] (5) After the defatted bovine heart tube fermentation broth is cooled to 55°C, add 25g (2.5% of the pretreated bovine heart tube mass) of alkaline protease for one enzymatic hydrolysis. The enzymatic hydrolysis pH is 7.0, the enzymatic hydrolysis time is 1h, and the enzyme is inactivated at 90°C for 30min. The alkaline protease is Bacillus licheniformis alkaline protease.

[0076] After enzyme inactivation was completed and the temperature dropped to 55℃, 10g (1.0% of the pretreated bovine heart tube mass) of trypsin was added for a second enzymatic hydrolysis. The hydrolysis pH was 7.5, the hydrolysis time was 3h, and the enzyme was inactivated at 85℃ for 25min.

[0077] (6) Add 70g of activated carbon, keep warm at 50℃ for 1h, then filter by plate and frame to obtain elastin peptide filtrate:

[0078] (7) The filtrate is treated with a 5000 Da ultrafiltration membrane to obtain a bovine heart tube elastin solution with a molecular weight of less than 5000 Da, and then spray-dried to obtain elastin peptides.

[0079] Example 3

[0080] (1) Take 2 kg of bovine heart tube, wash it and mince it, add 6 kg of 0.5% sodium bicarbonate solution, keep it at 50℃ for 0.5 h and then wash it with pure water;

[0081] (2) Add 6 kg of water to the cleaned beef heart tube mince, adjust the pH to 2 with hydrochloric acid, keep it at 70℃ for 3 hours, wash and dry it for later use.

[0082] (3) Take 1 kg of pretreated dried bovine heart tube fragments (80% protein content), add 10 kg of water for homogenization to obtain bovine heart tube homogenate, sterilize at 95℃ for 30 min, add 40 g of Lactobacillus paracasei YYS-K1 (CGMCC No.26405) and ferment at 39℃ for 18 h;

[0083] (4) After fermentation, remove the oil accumulated on the surface of the bovine heart tube fermentation liquid, sterilize at 95°C for 30 minutes, and obtain defatted bovine heart tube fermentation liquid after sterilization;

[0084] (5) After the defatted bovine heart tube fermentation broth is cooled to 55°C, 40g of alkaline protease (4% of the mass of the pretreated bovine heart tube) is added for the first enzymatic hydrolysis. The enzymatic hydrolysis pH is 9.0, the enzymatic hydrolysis time is 3h, and the enzyme is inactivated at 90°C for 30min. The alkaline protease is Bacillus licheniformis alkaline protease.

[0085] After enzyme inactivation is completed and the temperature is cooled to 55℃, add 5g (0.5% of the pretreated bovine heart tube mass) of trypsin for a second enzymatic hydrolysis. The hydrolysis pH is 6.5, the hydrolysis time is 1h, and the enzyme is inactivated at 85℃ for 30min.

[0086] (6) Add 80g of activated carbon, keep warm at 60℃ for 1h, then filter by plate and frame to obtain elastin peptide filtrate:

[0087] (7) The filtrate is treated with a 5000 Da ultrafiltration membrane to obtain a bovine heart tube elastin solution with a molecular weight of less than 5000 Da, and then spray-dried to obtain elastin peptides.

[0088] Among them, the Lactobacillus paracasei YYS-K1 used in Examples 1-3 is classified and named as: Lactobacillus paracasei ( Lactobacillus paracasei YYS-K1, Latin scientific name: Lacticaseibacillus paracasei The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 26405. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Date of deposit: January 6, 2023.

[0089] Comparative Example 1

[0090] The step of adding microbial inoculum for fermentation is removed; the remaining operations and processes are the same as in Example 1.

[0091] Comparative Example 2

[0092] Lactobacillus plantarum (using Lactobacillus plantarum ( Lactobacillus plantarum BMX2, deposited on September 6, 2018 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.16436, was used to replace the Lactobacillus paracasei (YYS-K1) strain used in fermentation. The remaining operations and processes were the same as in Example 1.

[0093] Comparative Example 3

[0094] The Lactobacillus paracasei (YYS-K1) strain used in fermentation was replaced with an equal amount of Lactobacillus plantarum (YYS-K3, deposited on June 12, 2023 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO. 27598). The remaining operations and processes were the same as in Example 1.

[0095] Comparative Example 4

[0096] The fermentation strain *Lactobacillus paracasei* (YYS-K1) was replaced in equal amounts with *Lactobacillus fermentum* (YYS-K2, deposited on April 17, 2023 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO. 27129). The remaining operations and processes were the same as in Example 1.

[0097] Comparative Example 5

[0098] The Lactobacillus paracasei strain (YYS-K1) used in fermentation was replaced with an equal amount of Lactobacillus rhamnosus (using Lactobacillus rhamnosus YYS-B2, which was deposited on August 14, 2023 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28164). The remaining operations and processes were the same as in Example 1.

[0099] Comparative Example 6

[0100] The enzymatic hydrolysis in step (3) of Example 1 was changed to a single enzymatic hydrolysis (omitting the trypsin hydrolysis step). The amount of alkaline protease added was 3% of the mass of the pretreated bovine heart tube, and the enzymatic hydrolysis time was 4 hours. The remaining operations and processes were the same as in Example 1.

[0101] Comparative Example 7

[0102] The enzymatic hydrolysis in step (3) of Example 1 was changed to a single enzymatic hydrolysis (omitting the alkaline protein hydrolysis step). The amount of trypsin added was 0.64% of the mass of the pretreated bovine heart tube, and the enzymatic hydrolysis time was 4 hours. The remaining operations and processes were the same as in Example 1.

[0103] Comparative Example 8

[0104] The enzymatic hydrolysis in step (3) is changed to the following: the amount of alkaline protease added for the first enzymatic hydrolysis is 3.0% of the mass of the pretreated bovine heart tube; the amount of papain (enzyme activity of 200,000 U / g, which is expressed as 200,000 U of enzyme activity per gram of enzyme) added for the second enzymatic hydrolysis is 0.32% of the mass of the pretreated bovine heart tube. The remaining operations and processes are the same as in Example 1.

[0105] In Example 1, the amount of trypsin added was 6.4g. Based on the protein content in the pretreated bovine heart tube, the enzyme addition amount of trypsin was 800U / g. The specific conversion process is as follows:

[0106] The enzyme activity of 6.4g of trypsin is: 100,000 U / g * 6.4 = 640,000 U. Converted to the amount of enzyme added per gram of pre-treated bovine heart tube, it is: 640,000 U / (1000g * 80%)g = 800 U / g.

[0107] Therefore, the alkaline protease activity of 200,000 U / g and the trypsin activity of 100,000 U / g mentioned in this article refer to the enzyme activity per gram of enzyme (used to characterize the enzyme's ability to catalyze a certain chemical reaction), which has a different meaning from the enzyme addition unit U / g obtained by converting the amount of protein in the pretreated bovine heart tube.

[0108] The conversion method for the addition of trypsin, papain, etc. in other comparative examples is the same as the calculation method above.

[0109] Therefore, the 3.2g papain in this comparative example, based on the protein content in the pretreated bovine heart tube, is 800U / g, which is the same amount as the trypsin used in Example 1.

[0110] Comparative Example 9

[0111] The enzymatic hydrolysis in step (3) of Example 1 was changed to a single enzymatic hydrolysis (omitting the trypsin hydrolysis step). The amount of alkaline protease added was 3.32% (33.2g) of the pretreated bovine heart tube mass, and the enzymatic hydrolysis time was 4h. The remaining operations and processes were the same as in Example 1.

[0112] In Example 1, the amount of alkaline protease added was 30g, which, based on the protein content in the pretreated bovine heart tube, represented an enzyme content of 7500 U / g. The amount of trypsin added was 6.4g, which, based on the protein content in the pretreated bovine heart tube, represented an enzyme content of 800 U / g. Therefore, the 33.2g of alkaline protease in this comparative example, based on the protein content in the pretreated bovine heart tube, represented 8300 U / g, which is the same as the total amount of trypsin and alkaline protease used in Example 1.

[0113] Comparative Example 10

[0114] The enzymatic hydrolysis in step (3) of Example 1 was changed to a single enzymatic hydrolysis (omitting the alkaline protein hydrolysis step). The amount of trypsin added was 6.64% (66.4g) of the pretreated bovine heart tube mass, and the enzymatic hydrolysis time was 4h. The remaining operations and processes were the same as in Example 1.

[0115] In Example 1, the amount of alkaline protease added was 30g, which, based on the protein content in the pretreated bovine heart tube, resulted in an enzyme content of 7500 U / g. The amount of trypsin added was 6.4g, which, based on the protein content in the pretreated bovine heart tube, resulted in an enzyme content of 800 U / g. Therefore, the 66.4g of trypsin in this comparative example, based on the protein content in the pretreated bovine heart tube, is 8300 U / g, which is the same as the total amount of trypsin and alkaline protease used in Example 1.

[0116] It should be noted that the enzymes with the same name used in the above embodiments and comparative examples are from the same commercially available company and brand, that is, the enzyme activity of the enzymes with the same name used in each embodiment and comparative example is consistent.

[0117] The performance of the elastin peptide products obtained in the above examples and comparative examples was tested:

[0118] Sensory effects of different bacterial strains fermenting bovine heart tube slurry on elastin peptides

[0119] (1) The elastin peptide powders from the examples and comparative examples were prepared into 10 mg / ml solutions (using pure water as the solvent). Their sensory characteristics were evaluated from two aspects: oil distribution, fishy smell, and sour taste. The sensory evaluation results are shown in Table 1.

[0120] Table 1 Sensory Analysis and Evaluation Table

[0121]

[0122] As can be seen from the comparison in Table 1,

[0123] Comparative Example 1, the unfermented bovine heart tube slurry, contained a lot of oil and had a strong fishy smell; Comparative Example 3, although the oil was more aggregated, had a strong odor; the oil in Comparative Examples 2, 4, and 5 was dispersed and was not considered (the oil was in the form of dispersed droplets, and the oil and peptide hydrolysate could not be removed by filtration equipment, while the oil in the examples was in the form of lumps, so the aggregated oil could be directly intercepted by a 10-20 mesh filter).

[0124] The fermented broth of bovine heart tubes obtained in Examples and Comparative Examples 6-10 not only has a good flavor, but its oil is also white and lumpy, and relatively aggregated. This indicates that fermentation with a specific Lactobacillus paracasei YYS-K1 (CGMCC No.26405) can help remove the oil, and the resulting elastin peptides have a better taste and flavor.

[0125] 2. Antioxidant activity of the elastin peptides prepared in the examples and comparative examples

[0126] The elastin peptide powders from Examples 1 and Comparative Examples 6-10 were prepared into a 20 mg / ml solution using water (pure water as the solvent). The superoxide anion scavenging rate, hydroxyl radical scavenging rate, DPPH radical scavenging rate, and ABTS radical scavenging rate were then tested. Detailed data are shown in Table 2.

[0127] The method for detecting the superoxide anion scavenging rate of elastin peptides was based on the study of the extraction process and antioxidant activity of Gleditsia sinensis leaf polysaccharides. The methods for detecting the DPPH and ABTS free radical scavenging rates were based on the DPPH and ABTS methods for antioxidant determination of elastin peptides in GB / T 39100~2020. The method for detecting the hydroxyl free radical scavenging rate was based on the study of antioxidant activity of Panax notoginseng polysaccharides.

[0128] Table 2. Antioxidant capacity of elastin peptides obtained by different enzymatic hydrolysis processes

[0129]

[0130] The test data shows that:

[0131] The examples all exhibit high scavenging rates against DPPH radicals, ABTS radicals, and hydroxyl radicals, demonstrating high antioxidant activity; among them, Example 1 showed scavenging rates of 100%, 84.27%, and 100.00% for hydroxyl radicals, DPPH radicals, and ABTS radicals, respectively.

[0132] The superoxide ion scavenging rate, DPPH radical scavenging rate and ABTS radical scavenging rate of the examples were all higher than those of Comparative Examples 1 and 6-10; the DPPH radical scavenging rate was the lowest in Comparative Example 6.

[0133] This indicates that the elastin peptides prepared by using Lactobacillus paracasei YYS-K1 fermentation treatment and a specific enzymatic hydrolysis combination process (alkaline protease + trypsin) in the embodiments of this application have superior antioxidant activity.

[0134] 3. Inhibitory activity of elastin peptides against pancreatic lipase and α-amylase

[0135] The elastin peptide powders of Examples 1 and Comparative Examples 6-10 were prepared into a 20 mg / ml solution with water (the solvent was pure water), and their pancreatic lipase inhibition rate and α-amylase inhibition rate were detected. The specific data are shown in Table 3.

[0136] The determination of elastin peptide pancreatic lipase inhibitory activity was performed in accordance with GB / T 23535-2009 National Standard: Lipase Preparations, and the determination of α-amylase inhibitory activity was performed in accordance with GB / T 24401~2009—Appendix B Mesophilic α-amylase Activity Determination Visual Colorimetric Method.

[0137] Table 3. Inhibition rates of pancreatic lipase and α-amylase in elastin peptides processed by different methods

[0138]

[0139] As can be seen from Table 3:

[0140] The examples showed inhibitory activity against pancreatic lipase and α-amylase, with inhibition rates of over 78% and 77%, respectively.

[0141] Comparative Example 1, the unfermented elastin peptides, showed no detectable pancreatic lipase inhibitory activity and an α-amylase inhibition rate of only 4%. Comparative Examples 6-10, while exhibiting pancreatic lipase and α-amylase inhibitory activities, showed significantly lower levels than the examples.

[0142] This application demonstrates that the fermentation treatment with Lactobacillus paracasei YYS-K1 (CGMCC No. 26405) and the combined enzymatic hydrolysis treatment with alkaline protease and trypsin can yield elastin peptides with superior inhibitory effects on pancreatic lipase and α-amylase.

[0143] 4. Glycosaminoglycan content of elastin peptides

[0144] The elastin peptide powders of Examples 1 and Comparative Examples 6-10 were prepared into a 20 mg / ml solution with water (the solvent was pure water), and their glycosaminoglycan content was detected. The specific data are shown in Table 4.

[0145] The determination of glycosaminoglycan content in elastin peptides was performed using the alcinolan colorimetric method, specifically referring to "Extraction, Separation and Bioactivity Study of Glycosaminoglycans from Scallop Skirt".

[0146] Table 4. Glycosaminoglycan content of elastin peptides processed by different methods

[0147]

[0148] As can be seen from Table 4:

[0149] The examples have high glycosaminoglycan content, all exceeding 8%, while the glycosaminoglycan content of Comparative Example 1 is only 4.43%. The examples in this application demonstrate that fermentation treatment with Lactobacillus paracasei YYS-K1 (CGMCC No. 26405) can significantly increase the glycosaminoglycan content in elastin peptides.

[0150] Furthermore, the glycosaminoglycan content of alkaline protease monoenzyme hydrolysis in Comparative Example 6 was 4.35%, while the glycosaminoglycan content of alkaline protease hydrolysis in Comparative Example 7 was only 3.85%. Although the glycosaminoglycan content of Comparative Examples 9-10 reached over 6%, it was lower than that of the example. This shows that under the same enzyme activity, the combination of alkaline protease and trypsin is also superior to monoenzyme hydrolysis. This indicates that the glycosaminoglycan content in the elastin peptides obtained by using alkaline protease and trypsin dual-enzyme hydrolysis in this application is higher than that obtained by alkaline protease or trypsin monoenzyme hydrolysis.

[0151] Meanwhile, the glycosaminoglycan content of the combined enzymatic hydrolysis of alkaline protease and papain in Comparative Example 8 reached 6.91%, indicating that although combined enzymatic hydrolysis can increase the glycosaminoglycan content in elastin peptides, the combined enzymatic hydrolysis combination of alkaline protease and trypsin used in this application is more conducive to increasing the glycosaminoglycan content.

[0152] 5. Albumin denaturation inhibitory activity of elastin peptides

[0153] The elastin peptide powders from Examples 1 and Comparative Examples 6-10 were prepared into a 20 mg / ml solution with water (solvent was water), and their albumin denaturation inhibition rate was detected. Detailed data are shown in Table 5.

[0154] The determination of the elastin peptide albumin denaturation inhibitory activity was based on the study "In Vitro Anti-inflammatory and Antioxidant Activity of an Ayurvedic Formulation - Trayodashang Guggulu".

[0155] Table 5. Albumin denaturation inhibition rate of elastin peptides processed by different methods

[0156]

[0157] As can be seen from Table 5:

[0158] The elastin peptides prepared in Example 1 showed a high albumin denaturation inhibition rate, indicating that they have strong anti-inflammatory activity. The elastin peptides prepared in Comparative Example 1 without fermentation treatment had a slightly lower albumin denaturation inhibition rate than those in Example 1. The elastin peptides prepared in Comparative Examples 6-10 using other enzymatic hydrolysis processes had lower albumin denaturation inhibition rates than those in Example 1. This indicates that fermentation by Lactobacillus paracasei YYS-K1 (CGMCC No. 26405) and combined enzymatic hydrolysis by alkaline protease and trypsin have a synergistic effect, which can significantly improve the anti-inflammatory activity of elastin peptides.

[0159] 6. AGEs-inhibiting (anti-glycation) activity of elastin peptides

[0160] The elastin peptide powders of Examples 1 and Comparative Examples 6-10 were prepared into a 20 mg / ml solution with water (solvent was water), and their AGEs inhibition rate was detected. The specific data are shown in Table 6.

[0161] The determination of the inhibitory (anti-glycation) activity of elastin peptide AGEs was based on the study of the anti-glycation effect and active ingredients of Sophora japonica flower water extract by multi-model evaluation.

[0162] Table 6. AGEs inhibition rate of elastin peptides processed by different methods

[0163]

[0164] As can be seen from Table 6:

[0165] The elastin peptides prepared in the examples showed a high AGEs inhibition rate, indicating that they have strong anti-glycation activity.

[0166] The AGEs inhibition rate of the elastin peptides obtained in Comparative Example 1 without fermentation was significantly lower than that in the Example. The AGEs inhibition rate of the elastin peptides obtained in Comparative Examples 6-10 using other enzymatic hydrolysis processes was also significantly lower than that in the Example. This indicates that the combined enzymatic hydrolysis of Lactobacillus paracasei YYS-K1 (CGMCC No. 26405) and alkaline protease and trypsin has a synergistic effect, which enhances the anti-glycation activity of elastin peptides.

[0167] The AGEs inhibition rate of Alkaline protease monoenzyme hydrolysis in Comparative Example 6 was higher than that of trypsin monoenzyme hydrolysis in Comparative Example 7, but lower than that of the combined hydrolysis of Alkaline protease and papain in Comparative Example 8. Although Comparative Examples 9-10 had the same enzyme activity as the examples, the combination of Alkaline protease and trypsin used in the examples was also better than that of Comparative Examples 9-10. Note: Although combined hydrolysis can improve the anti-inflammatory activity of elastin peptides compared to monoenzyme hydrolysis, the combined hydrolysis of the specific Alkaline protease and trypsin used in this application is more conducive to improving the AGEs inhibition rate.

[0168] In summary, the method for preparing elastin peptides provided by this invention has at least the following innovative points, mechanisms of action, and technical effects:

[0169] Its innovative features:

[0170] The difference between this invention and existing research is that:

[0171] (1) Using specific strains of bacteria fermentation technology to gather the oil in the raw slurry of bovine heart tube, the oil can be removed more thoroughly and conveniently for actual production operations, while improving the poor flavor of elastin peptides.

[0172] (2) The elastin peptides obtained by fermentation and enzymatic hydrolysis using a specific combination of enzyme hydrolysis and fermentation technology have high glycosaminoglycan content and exhibit pancreatic lipase inhibitory activity, antioxidant activity, α-amylase inhibitory activity, anti-glycation and anti-inflammatory activity.

[0173] Technical effects:

[0174] (1) The method of the present invention uses Lactobacillus paracasei YYS-K1 to treat the homogenized liquid of bovine heart tube, and then sequentially treats it with a specific enzymatic combination (alkaline protease, trypsin) to obtain elastin peptides with high glycosaminoglycan content:

[0175] (2) The elastin peptide obtained by processing bovine heart tubes in this invention has α-amylase inhibitory activity, antioxidant activity, pancreatic lipase inhibitory activity, anti-glycation and anti-inflammatory activity, and can be used as a functional factor in functional products.

[0176] Among them, glycosaminoglycans have the following characteristics and functions: their strong hydrophilicity is important for maintaining moisture in loose connective tissue; glycosaminoglycans are polyvalent anions with a strong affinity for K, Na, Ca, Mg, etc., thus regulating the distribution of these ions in tissues; they have a high viscosity for hyaluronic acid, providing lubrication and protection when attached to joint surfaces; and they have biological functions such as promoting wound healing. Therefore, adding elastin peptides with high glycosaminoglycan content to functional products (such as cosmetics and skin care products) for application on the human skin surface can achieve functions such as moisturizing, lubrication, promoting wound healing, and delaying skin aging.

[0177] In addition, glycosaminoglycans (GAGs) have various biological activities such as lowering blood sugar, lowering blood lipids, and antioxidation, which makes this elastin peptide widely applicable in functional foods.

[0178] Inhibiting α-amylase in the human body can effectively suppress carbohydrate absorption, thus reducing obesity and lowering blood sugar levels. Based on the high α-amylase inhibitory activity of this elastin peptide, its application in functional products (such as food and health supplements) can achieve functions such as weight loss and blood sugar reduction.

[0179] Because free radicals are atoms or groups with highly reactive unpaired electrons, excessive free radicals and oxidants in the human body can cause various harms, including attacking body cells and breaking down body tissues. Based on the high antioxidant properties of this elastin peptide, its application in functional products (such as food and health supplements) can provide functions such as health maintenance and anti-aging.

[0180] Because it inhibits the activity of pancreatic lipase secreted by the human body, it can prevent the breakdown of lipids in the intestines. Based on the high pancreatic lipase inhibitory activity of this elastin peptide, it can be used in functional products (such as food and health products) to achieve functions such as weight loss.

[0181] Due to its anti-inflammatory and anti-glycation properties, this elastin peptide can be used in functional products, such as skincare products, to reduce skin inflammation, slow down skin glycation, and alleviate skin aging.

[0182] (3) The elastin peptides prepared by this invention have no obvious bitterness or sourness, and have a good taste and flavor, which is conducive to improving the user experience.

[0183] (4) The method of the present invention can obtain the required elastin peptides by simply combining operations such as mixing, fermentation, enzymatic hydrolysis and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0184] It should be noted that:

[0185] (1) Definition:

[0186] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0187] The term "food" as used herein is used in a broad sense, encompassing both human food and drink. In some embodiments, the food product is suitable for and designed for human consumption.

[0188] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0189] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0190] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0191] The term "ultrafiltration" as 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.

[0192] (2) Raw materials used in implementation:

[0193] The alkaline protease, trypsin, papain, and other enzymes used are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0194] (3) Applications of elastin peptides:

[0195] Elastin peptides possess the following characteristics: (1) they exhibit α-amylase inhibitory activity; (2) they possess antioxidant properties; (3) they exhibit pancreatic lipase inhibitory activity; (4) they possess anti-glycation activity; and (5) they possess anti-inflammatory activity. Based on these characteristics (1)-5), elastin peptides can also be used in functional products with specific effects such as skin care, weight loss, blood sugar reduction, anti-oxidation, anti-glycation, and anti-inflammation.

[0196] 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.

[0197] 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.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 elastin peptides, characterized in that... This includes the following steps: The crushed bovine heart tubes were pre-treated to obtain pre-treated bovine heart tubes, which were then mixed with water and homogenized to obtain a homogenized solution. The homogenized liquid was sterilized for the first time, and then Lactobacillus paracasei was added for fermentation to obtain a fermentation broth; wherein, the Lactobacillus paracasei was Lactobacillus paracasei YYS-K1, and its preservation number was CGMCC No.26405; Remove the grease that has accumulated on the surface of the fermentation broth and perform a second sterilization treatment to obtain a defatted fermentation broth; Alkaline protease was added to the defatted fermentation broth to adjust the pH to 7-9, and the mixture was hydrolyzed at 50-55°C for 1-3 hours, followed by enzyme inactivation treatment at 85-90°C for 20-30 minutes to obtain the first hydrolysate. Trypsin was added to the first hydrolysate to adjust the pH to 6.5-7.5, and the mixture was hydrolyzed at 50-55°C for 1-3 hours, followed by enzyme inactivation treatment at 85-90°C for 20-30 minutes to obtain the second hydrolysate. The amount of alkaline protease added was 6250-10000 U / g, based on the protein content per gram of pretreated bovine heart tube; the amount of trypsin added was 625-1250 U / g, based on the protein content per gram of pretreated bovine heart tube. Activated carbon was added to the second hydrolysate after enzyme inactivation treatment, followed by filtration to obtain elastin peptide filtrate. The filtrate is subjected to ultrafiltration to retain elastin peptides with a molecular weight of less than 5000 Da, and then spray-dried to obtain the elastin peptides.

2. The method for preparing elastin peptides according to claim 1, characterized in that: After pretreatment, the crushed bovine heart tubes are mixed with water and homogenized. The preprocessing process is as follows: A 0.3%–0.5% sodium bicarbonate solution was added to the crushed bovine heart tubes, and the mixture was kept at 50℃–70℃ for 0.5–2 hours. Solid-liquid separation was then performed to obtain the bovine heart tube solid material. The mass ratio of the crushed bovine heart tubes to the sodium bicarbonate solution was 1:(2–4). Water is added to the bovine heart tube solid material, and the pH of the system is adjusted to 2-4 using hydrochloric acid solution. After being kept at 70℃-90℃ for 2-4 hours, solid-liquid separation is performed, and the material is dried to obtain the pretreated bovine heart tube. The mass ratio of the bovine heart tube solid material to water is 1:(2-4).

3. The method for preparing elastin peptides according to claim 2, characterized in that: During the homogenization process, the mass ratio of the pretreated bovine heart tube to the water is 1:(5-10).

4. The method for preparing elastin peptides according to claim 2, characterized in that: The mass of the Lactobacillus paracasei is (2-4) of the mass of the bovine heart tube after pretreatment.

5. The method for preparing elastin peptides according to claim 1, characterized in that: During the fermentation process, the fermentation temperature is 35–39°C and the fermentation time is 18–30 hours.

6. The method for preparing elastin peptides according to claim 1, characterized in that: During the treatment process, the activated carbon is added at a mass of 7-8% of the pretreated bovine heart tube mass, the treatment temperature is 50-60℃, and the treatment time is 0.5-1.5h.

7. An elastin peptide, characterized in that: The elastin peptide was prepared using the method described in any one of claims 1-6.

8. A functional product, characterized in that: Its components include elastin peptides prepared by the preparation method according to any one of claims 1-6.

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