A type of tendon peptide with antioxidant and pancreatic lipase inhibitory activities and its preparation method

Abstract

This invention relates to the technical field of deep processing of sturgeon by-products, and particularly to a sturgeon tendon peptide with antioxidant and pancreatic lipase inhibitory activities and its preparation method. The preparation method includes the following steps: mixing sturgeon with water and crushing it to form a first slurry; adding Bacillus licheniformis alkaline protease to the first slurry for a first hydrolysis; adding Bacillus subtilis neutral protease to the first hydrolysate for a second hydrolysis; adding a complex enzyme to the second hydrolysate for a third hydrolysis to obtain a sturgeon tendon hydrolysate; filtering the sturgeon tendon hydrolysate to remove impurities to obtain a filtrate, which is then filtered again to retain polypeptides, thus obtaining the sturgeon tendon peptide; wherein the complex enzyme includes pectinase and amylase. Using this method, a sturgeon tendon peptide with high glycosaminoglycan content can be obtained, which exhibits pancreatic lipase inhibitory activity, antioxidant activity, Staphylococcus aureus inhibitory activity, and prebiotic effects that promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

Description

Technical Field

[0001] This invention relates to the technical field of deep processing of sturgeon by-products, and in particular to a tendon peptide with antioxidant and pancreatic lipase inhibitory activities and its preparation method. Background Technology

[0002] The spinal cord of a sturgeon is called the dragon tendon. Currently, my country has a huge sturgeon farming industry, with freshwater sturgeon production exceeding 100,000 tons as early as 2019. The farming, processing, and production of sturgeon generate a large number of byproducts such as heads, tails, fins, and cartilage.

[0003] Sturgeon tendon has been considered a delicacy since ancient times. The study "Nutritional Composition Analysis and Evaluation of Four Different Freeze-Dried Sturgeon Tendons" investigated different varieties of sturgeon tendon and concluded that they all possess high nutritional value. Sturgeon tendon has a high crude protein content, with freeze-dried products containing over 70% crude protein, making it an excellent raw material for extracting bioactive peptides. However, research on the deep processing of sturgeon tendon to prepare bioactive peptides is currently scarce.

[0004] The study "Isolation, Identification and Anticancer Mechanism of Active Peptides from the Tendon of Hybrid Sturgeon (Acipenseridae)" used single-enzyme hydrolysis to prepare active peptides from tendon, obtaining active peptides with anticancer activity. However, the products obtained by single-enzyme hydrolysis often have unpleasant flavors, such as obvious bitterness, fishiness, sourness and off-odors, resulting in poor taste.

[0005] In summary, currently, sturgeon spinal cord is mainly consumed fresh, and there are few reports on its deep processing; even fewer studies on extracting active peptides with good flavor and texture from it. Existing literature and patents do not report on the preparation of peptides with antioxidant, antibacterial, and prebiotic-promoting activities from sturgeon spinal cord. Summary of the Invention

[0006] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing tendon peptides with antioxidant and pancreatic lipase inhibitory activities, the technical solution of which is as follows:

[0007] The preparation method of this tendon peptide includes the following steps:

[0008] The tendons are mixed with water and then crushed to form the first slurry.

[0009] Bacillus licheniformis alkaline protease was added to the first slurry to carry out the first hydrolysis, and the first hydrolysate was obtained.

[0010] Bacillus subtilis neutral protease was added to the first hydrolysate for a second hydrolysis to obtain the second hydrolysate.

[0011] A complex enzyme was added to the second hydrolysate for a third hydrolysis to obtain the tendon hydrolysate.

[0012] After filtering the hydrolysate of the tendon to remove impurities, the filtrate is obtained. The filtrate is then filtered to retain the polypeptide, thus obtaining the tendon peptide.

[0013] The complex enzyme includes pectinase and amylase.

[0014] In one embodiment, the filtrate is filtered to retain peptides with a molecular weight cutoff of less than 5000 Da. In another embodiment, the filtrate is ultrafiltered through an ultrafiltration membrane to retain peptides with a molecular weight cutoff of less than 5000 Da.

[0015] In one embodiment, the hydrolysate of the tendon is filtered using a plate and frame filter to remove insoluble substances, yielding a filtrate.

[0016] In one embodiment, the amount of Bacillus licheniformis alkaline protease added is 1-1.5% of the mass of the tendon; the amount of Bacillus subtilis neutral protease added is 0.3-0.5% of the mass of the tendon; and the amount of the compound enzyme added is 1-3% of the mass of the tendon, wherein the mass ratio of pectinase to amylase is 1:(0.8-1.2).

[0017] In one embodiment, during the first hydrolysis, the pH of the first slurry is adjusted to 7.0–7.5, the constant temperature hydrolysis temperature is 55±1℃, and the constant temperature hydrolysis time is 100–240 min; during the second hydrolysis, the constant temperature hydrolysis temperature is 55±1℃, and the constant temperature hydrolysis time is 100–240 min; during the third hydrolysis, the constant temperature hydrolysis temperature is 55±1℃, and the constant temperature hydrolysis time is 40–80 min.

[0018] In one embodiment, the pH of the first slurry is adjusted with hydrochloric acid.

[0019] In one embodiment, after the first hydrolysis, the enzyme is first inactivated before a second hydrolysis is performed; after the second hydrolysis, the enzyme is first inactivated before a third hydrolysis is performed; and after the third hydrolysis, the enzyme is first inactivated before filtration to remove impurities.

[0020] In one embodiment, after the first hydrolysis, the first hydrolysate is heated to 80-85°C to inactivate the enzyme for 20-30 minutes; the second hydrolysate is heated to 80-85°C to inactivate the enzyme for 20-30 minutes; the third hydrolysate is heated to 85-90°C to inactivate the enzyme for 20-30 minutes, and then cooled to room temperature.

[0021] In one embodiment, the tendon is added to water and crushed using a colloid mill to form a first slurry; wherein the mass ratio of the tendon to water is 1:(5-10).

[0022] The present invention also provides a tendon peptide, which is prepared by the tendon peptide preparation method described above.

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

[0024] In one embodiment, the functional product includes functional foods, health products, cosmetics, and skincare products; and the functional product includes at least one of the following characteristics:

[0025] (1) It possesses antioxidant properties;

[0026] (2) It possesses pancreatic lipase inhibitory activity;

[0027] (3) It possesses inhibitory activity against Staphylococcus aureus;

[0028] (4) It has beneficial bacteria promoting activity.

[0029] Based on the above, compared with the prior art, the preparation method of the tendon peptide of the present invention has the following beneficial effects:

[0030] The method of this invention can be used to prepare tendon peptides with high glycosaminoglycan content. These tendon peptides have pancreatic lipase inhibitory activity, antioxidant activity, Staphylococcus aureus inhibitory activity, and prebiotic effects that promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0031] This dragon tendon peptide is prepared by hydrolysis using a combination of various enzymatic hydrolysates. It has no obvious bitter, fishy, ​​sour, or off-odor taste. When applied to food, it has a good taste and flavor, which helps to improve the user experience.

[0032] The method of this invention can produce the desired peptides by simply combining operations such as mixing, enzymatic hydrolysis, and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0033] 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 and other beneficial effects of the invention can be realized and obtained by means of the structures particularly pointed out in the description, claims and drawings. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0035] Figure 1 A graph showing the content of glycosaminoglycans in peptide samples prepared under different processing conditions;

[0036] Figure 2 This is a graph showing the antioxidant capacity of peptide samples prepared under different processing conditions. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0038] 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.

[0039] This invention provides an operational example of a method for preparing tendon peptides, the specific steps of which are as follows:

[0040] (1) Add the tendons to room temperature pure water at a mass ratio of 1: (5-10), crush them with a colloid mill, and make the first slurry.

[0041] (2) Heat to 55±1℃, adjust the pH of the first slurry to 7.0~7.5 with hydrochloric acid, add 1~1.5% of the raw material mass of Bacillus licheniformis alkaline protease, hydrolyze for 100~240min; heat to 80~85℃ to inactivate enzyme for 20~30min.

[0042] (3) After cooling to 55±1℃, add Bacillus subtilis neutral protease at 0.3~0.5% of the raw material mass, hydrolyze for 100~240min, and then heat to 80~85℃ to inactivate the enzyme for 20~30min.

[0043] (4) After cooling to 55±1℃, add compound enzyme at 1-3% of the raw material mass. After enzymatic hydrolysis for 40-80 min, heat to 85-90℃ to inactivate the enzyme for 20-30 min, and then cool to room temperature.

[0044] The complex enzyme is composed of pectinase and amylase, and the mass ratio of pectinase to amylase is 1:(0.8-1.2).

[0045] (5) The hydrolysate of the tendon was filtered by plate and frame filter to remove insoluble substances and obtain filtrate; the filtrate was ultrafiltered through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight of less than 5000 Da.

[0046] (6) Dry the tendon peptide into powder and test the glycosaminoglycan content; spray drying can be used for the powder drying process.

[0047] (7) The activity of the dragon tendon peptide was tested and it was found to have antioxidant activity, pancreatic lipase inhibitory activity, Staphylococcus aureus inhibitory activity and beneficial bacteria promoting activity.

[0048] The present invention provides the following embodiments and comparative examples.

[0049] Example 1

[0050] 1. Take 100g of dragon tendon, mix it with 1000g of room temperature pure water, and crush it using a colloid mill.

[0051] 2. Heat the system to 55℃, adjust the pH to 7.0 with hydrochloric acid, add 1g of Bacillus licheniformis alkaline protease to hydrolyze for 240min; heat to 85℃ to inactivate the enzyme for 30min.

[0052] 3. After cooling to 55℃, add 0.3g of Bacillus subtilis neutral protease, hydrolyze for 240min, and then heat to above 85℃ for 30min to inactivate the enzyme.

[0053] 4. After cooling to 55℃, add 1g of compound enzyme and hydrolyze for 40min. After hydrolysis, heat to 90℃ to inactivate the enzyme for 20min. The mass ratio of pectinase to amylase in the compound enzyme is 1:1.2.

[0054] 5. The hydrolysate of the tendon was filtered through a plate and frame filter to remove insoluble substances, yielding a filtrate. The filtrate was then ultrafiltered through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight less than 5000 Da.

[0055] 6. Store in powder form using spray drying.

[0056] Example 2

[0057] 1. Take 100g of dragon tendon, mix it with 800g of room temperature pure water, and crush it using a colloid mill.

[0058] 2. Heat the system to 56°C, adjust the pH to 7.2 with sodium hydroxide, add 1.5g of Bacillus licheniformis alkaline protease to hydrolyze for 120min; heat to 85°C to inactivate the enzyme for 25min.

[0059] 3. After cooling to 56℃, add 0.5g of Bacillus subtilis neutral protease, hydrolyze for 120min, and then heat to above 85℃ for 25min to inactivate the enzyme.

[0060] 4. After cooling to 56℃, add 1.5g of compound enzyme and hydrolyze for 40min. After hydrolysis, heat to 90℃ to inactivate the enzyme for 25min. The mass ratio of pectinase to amylase in the compound enzyme is 1:0.8.

[0061] 5. The hydrolysate of the tendon was filtered through a plate and frame filter to remove insoluble substances, yielding a filtrate. The filtrate was then ultrafiltered through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight less than 5000 Da.

[0062] 6. Store in powder form using spray drying.

[0063] Example 3

[0064] 1. Take 100g of dragon tendon and mix it with 500g of room temperature pure water, then crush it using a colloid mill.

[0065] 2. Heat the system to 56°C, adjust the pH to 7.2 with sodium hydroxide, add 1.5g of Bacillus licheniformis alkaline protease to hydrolyze for 100min; heat to 85°C to inactivate the enzyme for 20min.

[0066] 3. After cooling to 56℃, add 0.5g of Bacillus subtilis neutral protease, hydrolyze for 100min, and then heat to above 85℃ for 20min to inactivate the enzyme.

[0067] 4. After cooling to 56℃, add 1.5g of compound enzyme and hydrolyze for 60min. After hydrolysis, heat to 90℃ to inactivate the enzyme for 25min. The mass ratio of pectinase to amylase in the compound enzyme is 1:0.8.

[0068] 5. The hydrolysate of the tendon was filtered through a plate and frame filter to remove insoluble substances, yielding a filtrate. The filtrate was then ultrafiltered through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight less than 5000 Da.

[0069] 6. Store in powder form using spray drying.

[0070] Example 4

[0071] 1. Take 100g of dragon tendon, mix it with 500g of room temperature pure water, and crush it using a colloid mill.

[0072] 2. Heat the system to 56°C, adjust the pH to 7.5 with sodium hydroxide, add 1.2g of Bacillus licheniformis alkaline protease to hydrolyze for 200min; heat to 85°C to inactivate the enzyme for 30min.

[0073] 3. After cooling to 55℃, add 0.5g of Bacillus subtilis neutral protease, hydrolyze for 200min, and then heat to above 85℃ for 30min to inactivate the enzyme.

[0074] 4. After cooling to 56℃, add 1.2g of compound enzyme and hydrolyze for 80min. After hydrolysis, heat to 90℃ to inactivate the enzyme for 30min. The mass ratio of pectinase to amylase in the compound enzyme is 1:1.

[0075] 5. The hydrolysate of the tendon was filtered through a plate and frame filter to remove insoluble substances, yielding a filtrate. The filtrate was then ultrafiltered through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight less than 5000 Da.

[0076] 6. Store in powder form using spray drying.

[0077] Example 5

[0078] 1. Take 100g of dragon tendon, mix it with 1000g of room temperature pure water, and crush it using a colloid mill.

[0079] 2. Heat the system to 56°C, adjust the pH to 7.0 with sodium hydroxide, add 1g of Bacillus licheniformis alkaline protease to hydrolyze for 120min; heat to 85°C to inactivate the enzyme for 20min.

[0080] 3. After cooling to 54℃, add 0.3g of Bacillus subtilis neutral protease, hydrolyze for 120min, and then heat to above 85℃ to inactivate the enzyme for 20min.

[0081] 4. After cooling to 56℃, add 1.5g of compound enzyme and hydrolyze for 80min. After hydrolysis, heat to 90℃ to inactivate the enzyme for 20min. The mass ratio of pectinase to amylase in the compound enzyme is 1:1.

[0082] 5. Filter the hydrolysate of the tendon using a plate and frame filter to remove insoluble substances, obtaining the filtrate. Then, ultrafilter the filtrate through a 5000 Da ultrafiltration membrane to obtain tendon peptides with a molecular weight less than 5000 Da.

[0083] 6. Store in powder form using spray drying.

[0084] Comparative Example 1

[0085] Step 4 (i.e., canceling the use of compound enzyme to treat the tendon hydrolysate) is omitted, and the second hydrolysate is directly filtered to remove impurities. The remaining steps and processes are the same as in Example 1.

[0086] Comparative Example 2

[0087] In step 4, the compound enzyme was replaced with an equal amount of amylase for post-treatment (i.e., the amount of amylase used in Comparative Example 2 was equal to the amount of compound enzyme used), and everything else was the same as in Example 1.

[0088] Comparative Example 3

[0089] In step 4, the compound enzyme was replaced with an equal amount of pectinase for post-treatment (i.e., the amount of pectinase used in Comparative Example 3 was equal to the amount of compound enzyme used), and everything else was the same as in Example 1.

[0090] Comparative Example 4

[0091] In step 2, the pH was adjusted to 8.0, and the Bacillus licheniformis alkaline protease was replaced with an equal amount of trypsin. Everything else was the same as in Example 1.

[0092] Comparative Example 5

[0093] In step 2, papain was used to replace Bacillus licheniformis alkaline protease in an equal amount, and everything else was the same as in Example 1.

[0094] Comparative Example 6

[0095] In step 2, the pH was adjusted to 2.0, and the Bacillus licheniformis alkaline protease was replaced with an equal amount of pepsin; in step 3, the pH was adjusted to 7.0 before enzymatic hydrolysis, and everything else was the same as in Example 1.

[0096] Comparative Example 7

[0097] In step 2, the pH value was adjusted to 3.0, and the Bacillus licheniformis alkaline protease was replaced with an equal amount of Aspergillus niger acidic protease; in step 3, the pH value was adjusted to 7.0 before enzymatic hydrolysis, and everything else was the same as in Example 1.

[0098] It should be noted that: because the enzymes used in the above-mentioned (e.g., Comparative Examples 4, 6-7) are different from the enzymes originally used in the embodiments of this application, the pH of the solutions in the comparative examples is also adjusted adaptively in the enzyme replacement step to adapt to the use of different enzymes. In addition, since the pH of the previous step may affect the use of the enzyme in the next step, the pH is also adjusted before the next enzymatic hydrolysis step; for example, in step 2 of Comparative Example 7, since an enzyme suitable for use in an acidic environment is used, the pH is adjusted to acidic, and since the enzyme used in the next step is Bacillus subtilis neutral protease, which is suitable for a neutral environment, the pH needs to be adjusted to neutral again. The limiting role of the environmental pH value is to adapt to the use conditions of the enzyme, and is not a variable to be reflected in the design of the comparative examples in this paper.

[0099] The performance of the peptide products obtained in the above examples and comparative examples was tested:

[0100] 1. Effects of different processing conditions on the glycosaminoglycan content of peptide products

[0101] (1) Glycosaminoglycan detection was performed on the peptide samples of the examples and comparative examples, and the results are as follows: Figure 1 As shown. The method for detecting glycosaminoglycans was based on the method described in "Extraction Methods and Activity Comparison of Glycosaminoglycans from Deer Antler".

[0102] (2) Through detection data ( Figure 1 It can be seen that:

[0103] The product of Example 1 had the highest glycosaminoglycan content, reaching 5.6%.

[0104] The comparison results between Example 1 and Comparative Examples 1-3 show that:

[0105] Comparative Example 1 did not use a complex enzyme composed of pectinase and amylase for post-treatment, but only used protease for the first and second hydrolysis, and could not obtain a polypeptide product with a high content of glycosaminoglycans.

[0106] In Comparative Example 2, the content of glycosaminoglycans in the product was not increased compared to that in Comparative Example 1 when the complex enzyme was replaced with an equal amount of amylase.

[0107] Comparative Example 3 replaced the compound enzyme with an equal amount of pectinase. Compared with Comparative Example 1, the content of glycosaminoglycans in its product was increased, but compared with the example using the compound enzyme, the content of glycosaminoglycans was significantly reduced.

[0108] The reasons for this may be: pectinase and amylase have different enzymatic hydrolysis characteristics; the simultaneous use of pectinase and amylase can significantly increase the content of glycosaminoglycans, possibly due to their synergistic effect, which helps them to exert their enzymatic hydrolysis characteristics.

[0109] Comparative results of Example 1 and Comparative Examples 4-7 show that the glycosaminoglycan content prepared by different enzymatic hydrolysis combinations is different:

[0110] The example prepared by sequential hydrolysis of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and a complex enzyme (pectinase and amylase) yielded the highest glycosaminoglycan content in the tendon peptide.

[0111] In contrast, Comparative Examples 4-7 used other types of enzymes to replace the alkaline protease of Bacillus licheniformis (i.e., other enzymatic combinations), and the glycosaminoglycan content of the products was lower than that of the examples.

[0112] The reason for this may be that the specific enzymatic combination in the example is more conducive to the hydrolysis of tendon protein and the release of glycosaminoglycans.

[0113] 2. Effects of different process conditions on pancreatic lipase inhibition rate

[0114] (1) Prepare a 20 mg / ml solution (solvent is water) of the polypeptide powders of the examples and comparative examples, and detect the pancreatic lipase inhibition rate. See Table 1 for specific data. The detection method is in accordance with the study on the inhibitory effect of lotus leaf flavonoids on pancreatic lipase.

[0115] Table 1. Inhibition rate of pancreatic lipase in peptide samples processed by different methods

[0116]

[0117] Note: - indicates not detected.

[0118] (2) As can be seen from the data in Table 1:

[0119] Examples 1-5 all showed good pancreatic lipase inhibition rates, all above 78%; Example 2 showed a relatively good pancreatic lipase inhibition rate of 81.62%.

[0120] The examples, compared with Comparative Examples 4-7, show that different combinations of protease digestion can significantly affect the pancreatic lipase inhibitory activity of the obtained peptides:

[0121] No pancreatic lipase inhibitory activity was detected in Comparative Example 1 (without using a compound enzyme) and Comparative Example 2 (using only amylase). This may be because some glycoprotein polymers were still present in the tendon peptides prepared in Comparative Examples 1 and 2, resulting in insufficient proteolysis and failure to release effective active peptides.

[0122] Comparative Example 3, which was post-treated with pectinase only, showed some pancreatic lipase inhibitory activity, but it was significantly weaker than that of the Example. It is speculated that this may be because during the hydrolysis of the complex enzyme, after the amylase enzymatically hydrolyzes some of the glucan chains in the glycoprotein, the glycoprotein structure expands, and the pectinase can better enzymatically hydrolyze specific regions to produce a higher content of effective peptides. This is enough to show that there is a synergistic relationship between pectinase and amylase.

[0123] No pancreatic lipase inhibitory activity was detected in Comparative Examples 4-7, indicating that replacing the enzymatic digestion combination of Bacillus licheniformis alkaline protease with other types of enzymes cannot yield peptides with pancreatic lipase inhibitory activity.

[0124] 3. Effects on antioxidant activity

[0125] (1) The peptide powder samples from the examples and comparative examples were prepared into a 20 mg / ml solution (using water as the solvent), and their DPPH radical scavenging rate, ABTS radical scavenging rate, hydroxyl radical scavenging rate, and superoxide anion scavenging rate were detected. Detailed detection data can be found in [link to relevant documentation]. Figure 2 .

[0126] The detection methods for DPPH free radical scavenging rate, ABTS free radical scavenging rate, and hydroxyl free radical scavenging rate were based on "Isolation and characterization of three antioxidant peptides from protein hydrolysate of bluefin leatherjacket", and the detection method for superoxide anion scavenging rate was based on "Two novel antioxidant nonapeptides from protein hydrolysate of skate (Raja porosa) muscle".

[0127] (2) Through detection data ( Figure 2 It can be seen that:

[0128] The examples show high scavenging rates for DPPH radicals, ABTS radicals, hydroxyl radicals, and superoxide anions, demonstrating high antioxidant activity; among them, Example 3 shows DPPH radical scavenging rates and superoxide anion scavenging rates as high as 90.85% and 91.33%, respectively.

[0129] The DPPH radical scavenging rate and superoxide anion scavenging rate of Comparative Examples 1-7 were significantly lower than those of the Examples, with Comparative Example 3 showing the lowest DPPH radical scavenging rate and superoxide anion scavenging rate.

[0130] Although some comparative examples (e.g., comparative example 5) may have a certain free radical scavenging rate that is at or slightly higher than that of the examples, the overall antioxidant performance of the comparative examples is not as good as that of the examples. Their free radical scavenging rates are significantly lower than those of the examples. For example, the DPPH free radical scavenging rate and superoxide anion scavenging rate of comparative examples 1-7 are significantly lower than those of the examples, while the examples have high scavenging rates for DPPH free radicals, ABTS free radicals, hydroxyl free radicals and superoxide anions, and have the best overall performance.

[0131] 4. Effects on promoting the activity of beneficial bacteria

[0132] (1) The obtained dragon tendon peptide powder was used to detect the growth promotion rate of *Lactobacillus plantarum* and *Lactobacillus fermentum*. The specific results are shown in Table 2. The detection method was based on the study on the in vitro growth promotion effect of Chinese yam peptides on probiotics, with slight modifications.

[0133] The specific testing method is as follows:

[0134] MRS liquid culture medium was prepared and dispensed into 10 mL tubes. 300 μL of probiotic strains (Lactobacillus plantarum and Lactobacillus fermentum, respectively) were inoculated into each tube. The experimental group received 500 μL of 20 mg / mL ferrous sulfate aqueous solution, while the control group received 500 μL of sterile water. The cultures were incubated at 37°C for 24 h. The culture medium was then diluted 20-fold before OD was measured. 600 Calculate the probiotic promotion rate.

[0135] Probiotic promotion rate % = (BA) / A x 100%;

[0136] Note: A: OD of the blank group 600 B: OD of the experimental group 600 .

[0137] The MRS liquid culture medium formula is as follows: 10.0g casein digest, 10.0g beef extract, 5.0g yeast extract, 2.0g triammonium citrate, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 1.08g Tween-80, 0.2g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·4H2O), and distilled water to a final volume of 1000mL, pH 5.7–5.9.

[0138] Among them, Lactobacillus plantarum BXM2 was deposited at the China General Microbiological Culture Collection Center on September 6, 2018, with the accession number CGMCC NO.16436.

[0139] The fermenting Lactobacillus strain used was Lactobacillus fermentum B153, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 10, 2018, with accession number CGMCC No. 16454. Technicians can obtain it from the collection center using this accession number.

[0140] Table 2. Probiotic promotion rate of peptide samples from different processes

[0141]

[0142] Note: - indicates not detected, which means that it has no effect on promoting the growth of beneficial bacteria. In other words, compared with the control group, it does not promote the growth of beneficial bacteria, that is, its promotion rate is less than or equal to 0.

[0143] (2) As can be seen from the data in Table 2:

[0144] The examples demonstrated good beneficial bacteria-promoting activity, with *Lactobacillus plantarum* promoting proliferation at a rate of over 15% and *Lactobacillus fermentum* promoting proliferation at a rate of over 9%.

[0145] Comparative Examples 1, 3, 4, 5, 6, and 7 did not show any activity promoting beneficial bacteria.

[0146] Comparative Example 2 showed promoting activity, but it was significantly lower than that of the Comparative Example.

[0147] This indicates that the enzymatic combination of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and complex enzymes (pectinase and amylase) can produce polypeptides with probiotic activity.

[0148] 5. Effects on inhibiting Staphylococcus aureus

[0149] (1) The inhibition rate of Staphylococcus aureus was detected by using the plate count method. The results are detailed in Table 3.

[0150] The specific testing method is as follows:

[0151] Culture medium preparation: For the blank control group, use NB solid culture medium (without added peptide sample). For the experimental group, add 0.2% of the test sample powder to the solid culture medium, mix thoroughly, sterilize, and pour into plates for later use. The amount of test sample added is 2 g / L solid.

[0152] Blank control group NB solid culture medium formula: peptone 10.0g, beef extract powder 3.0g, sodium chloride 5.0g, agar 15.0g, distilled water to a final volume of 1000mL, pH 7.2~7.6.

[0153] The experimental group solid culture medium formula was as follows: 10.0g peptone, 3.0g beef extract powder, 5.0g sodium chloride, 15.0g agar, 2.0g draconide, and 1000mL distilled water, pH 7.2-7.6.

[0154] After activation, Staphylococcus aureus was diluted to 10⁻⁶ using a serial dilution method. -1 - 10 -7 For each concentration gradient, 100 μL of bacterial suspension at each concentration was evenly spread onto the solid culture medium of the blank control group and the solid culture medium containing the test sample. The plates were incubated upside down at 37°C for 24 hours. After incubation, the colonies on the plates were counted, ensuring the colony count was between 30 and 300. Plates with excessively high or low concentrations were discarded.

[0155] The formula for calculating the Staphylococcus aureus inhibition rate is as follows:

[0156] Staphylococcus aureus inhibition rate % = (AB) / A x 100%;

[0157] Note: A: Colony count in the blank control group; B: Colony count in the experimental group;

[0158] Table 3. Staphylococcus aureus inhibition rate of peptide samples processed by different methods

[0159]

[0160] Note: - indicates not detected, which means that it has no inhibitory effect on the proliferation of Staphylococcus aureus. In other words, compared with the control group, it does not inhibit the proliferation of Staphylococcus aureus, and its inhibition rate is less than or equal to 0.

[0161] (3) As can be seen from Table 3:

[0162] The examples showed inhibitory activity against Staphylococcus aureus, with an inhibition rate of over 18%.

[0163] Comparative Examples 1, 3, 4, 5, 6, and 7 did not show any inhibitory activity against Staphylococcus aureus;

[0164] Comparative Example 2 showed inhibitory activity against Staphylococcus aureus, but its inhibition rate was significantly lower than that of the examples.

[0165] This indicates that the enzymatic combination of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, and complex enzymes (pectinase and amylase) can also produce polypeptides with inhibitory activity against harmful bacteria.

[0166] 6. Effects of different processing conditions on sensory evaluation of peptide products

[0167] (1) The dragon tendon peptide powder of Examples 1-5 and Comparative Examples 1-7 was prepared into a 3% solution (the solvent was pure water). Its sensory characteristics were evaluated from four aspects: fishy taste, bitter taste, sour taste and off-odor. The sensory evaluation criteria are shown in Table 4.

[0168] Table 4 Sensory Evaluation Criteria

[0169]

[0170] The sample solutions were evaluated according to the evaluation criteria in Table 4, and the results are shown in Table 5.

[0171] Table 5 Sensory evaluation results of peptide solutions from various processes

[0172]

[0173] (2) From Table 5, we can obtain:

[0174] The flavor and taste of Comparative Examples 4-7 were significantly worse than those of the Examples, indicating that different combinations of protease hydrolysis (using other types of enzymes to replace Bacillus licheniformis alkaline protease) have a significant impact on the sensory properties of dragon tendon peptides, affecting their fishy, ​​bitter, sour, and off-odors to varying degrees.

[0175] Compared to the examples, the flavor and texture of Comparative Examples 1-3 are similar to those of the examples, indicating that the use of the combined enzymes of amylase and pectinase has no significant effect on the flavor of the tendon peptide.

[0176] The reason for this may be that the enzymatic hydrolysis process breaks down large protein molecules into smaller peptides or free amino acids, while simultaneously promoting the release of various intracellular substances, all of which affect the product's taste and aroma. The combination of Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease yields the best taste for the "dragon tendon peptide," with little or no fishy, ​​bitter, sour, or off-odor characteristics. Other protease combinations all exhibited noticeable unpleasant flavors.

[0177] In summary, the method for preparing tendon peptides provided by this invention has at least the following mechanisms of action and technical effects:

[0178] (1) The method of the present invention sequentially treats Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, pectinase and amylase to obtain tendon peptide with high glycosaminoglycan content:

[0179] (2) This invention is the first to discover that the sturgeon tendon peptide prepared by enzymatic hydrolysis of Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, pectinase and amylase has high pancreatic lipase inhibitory activity, high antioxidant activity (high DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, ABTS free radical scavenging rate, superoxide anion free radical scavenging rate), inhibits Staphylococcus aureus activity and promotes the activity of beneficial bacteria (Lactobacillus plantarum and Lactobacillus fermentum), and can be used as a functional factor in functional foods, cosmetics and health products.

[0180] Among them, glycosaminoglycans have the following characteristics and functions: their strong hydrophilicity is important for maintaining moisture in loose connective tissues; glycosaminoglycans are polyvalent anions with a strong affinity for K, Na, Ca, Mg, etc., thus they can regulate the distribution of these ions in tissues;

[0181] Because it inhibits the activity of intestinal lipase, it can prevent the breakdown of lipids in the intestine. Based on the high pancreatic lipase inhibitory activity of this active peptide, it can be applied in functional products (such as food and health products).

[0182] Since Staphylococcus aureus is a harmful bacterium, Staphylococcus aureus infection can lead to a variety of diseases. Active peptides have an inhibitory effect on Staphylococcus aureus and can be used as an antibacterial agent in various products, such as food, hand sanitizer, and skin care products, to kill harmful bacteria.

[0183] Since the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum* is beneficial to intestinal digestion and defecation, based on the prebiotic effect of this dragon tendon peptide in promoting the proliferation of *Lactobacillus plantarum* and *Lactobacillus fermentum*, its application in functional products (such as food) can play a role in aiding digestion and defecation.

[0184] (3) The dragon tendon peptide is prepared by a combination of various enzymatic hydrolysis. It has no obvious bitter, fishy, ​​sour and off-odor taste. When it is applied to food, it has a good taste and flavor, which is conducive to improving the user experience.

[0185] (4) The method of the present invention can obtain the desired polypeptide by simply combining operations such as mixing, enzymatic hydrolysis and filtration. It has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production.

[0186] It should be noted that:

[0187] (1) Definition:

[0188] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0189] 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.

[0190] In this article, "beneficial bacteria promotion activity" refers to the ability to promote the proliferation of Lactobacillus plantarum and Lactobacillus fermentum.

[0191] In the text, "DPPH" stands for 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazine (free radical).

[0192] In the text, "ABTS" refers to the free radical 2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0193] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0194] 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.

[0195] (2) Raw materials used in implementation:

[0196] The enzymes used, such as Bacillus licheniformis alkaline protease, Bacillus subtilis neutral protease, pectinase, and amylase, are all commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0197] (3) Application of Longjin peptide:

[0198] Longjin peptide possesses the following characteristics: 1) antioxidant properties; 2) pancreatic lipase inhibitory activity; 3) Staphylococcus aureus inhibitory activity; 4) beneficial bacteria promoting activity; and 5) high glycosaminoglycan content. Based on these characteristics (1)-5), Longjin peptide can also be applied to functional products with skin-care and other identifiable effects (functional products can be food, health products, cosmetics, skin care products, bath products, cleaning products, etc., including any substance providing preventative and / or other beneficial effects), including but not limited to skin care and other identifiable effects.

[0199] 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.

[0200] 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.

[0201] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a tendon peptide with antioxidant and pancreatic lipase inhibitory activities, characterized in that... This includes the following steps: The tendons are added to water and crushed using a colloid mill to form a first slurry; wherein the mass ratio of the tendons to water is 1:(5-10); Bacillus licheniformis alkaline protease was added to the first slurry for a first hydrolysis. After a first enzyme inactivation treatment, a first hydrolysate was obtained. The amount of Bacillus licheniformis alkaline protease added was 1-1.5% of the mass of the tendon. In the first hydrolysis, the pH of the first slurry was adjusted to 7.0-7.5, the constant temperature hydrolysis temperature was 55±1℃, and the constant temperature hydrolysis time was 100-240 min. Bacillus subtilis neutral protease was added to the first hydrolysate for a second hydrolysis. After the second enzyme inactivation treatment, a second hydrolysate was obtained. The amount of Bacillus subtilis neutral protease added was 0.3-0.5% of the mass of the tendon. In the second hydrolysis, the constant temperature hydrolysis temperature was 55±1℃ and the constant temperature hydrolysis time was 100-240 min. A compound enzyme is added to the second hydrolysate for a third hydrolysis. After a third enzyme inactivation treatment, a tendon hydrolysate is obtained. The compound enzyme comprises pectinase and amylase. The amount of the compound enzyme added is 1-3% of the tendon mass, and the mass ratio of pectinase to amylase is 1:(0.8-1.2). In the third hydrolysis, the isothermal hydrolysis temperature is 55±1℃, and the isothermal hydrolysis time is 40-80 min. After filtering the hydrolysate of the tendon to remove impurities, the filtrate is obtained. The filtrate is then filtered to retain polypeptides with a molecular weight of less than 5000 Da, thus obtaining the tendon peptide.

2. The method for preparing dragon tendon peptide according to claim 1, characterized in that: The filtrate is then ultrafiltered through an ultrafiltration membrane to remove polypeptides with a molecular weight of less than 5000 Da, thus obtaining the final product.

3. A type of tendon peptide, characterized in that: The dragon tendon peptide is prepared by the method for preparing dragon tendon peptide as described in any one of claims 1-2.

4. A functional product, characterized in that: Its components include the tendon peptide prepared by the preparation method according to any one of claims 1-2.

Images