Phosphorus shrimp active peptide and preparation method thereof, and functional food

Abstract

The present application relates to the technical field of defatted phosphor shrimp processing, and particularly relates to a phosphor shrimp active peptide, a preparation method thereof and a functional food. The preparation method comprises the following steps: defatted phosphor shrimp powder and water are used to form a first material liquid; Aspergillus oryzae acid protease and pepsin are added into the first material liquid for first hydrolysis, enzyme inactivation treatment and separation treatment to obtain supernatant A and precipitate B; the supernatant A is subjected to membrane filtration to intercept polypeptide A; the precipitate B and water are used to form a second material liquid; ficin is added into the second material liquid for second hydrolysis, enzyme inactivation treatment and separation treatment to obtain supernatant B; the supernatant B is subjected to membrane filtration to intercept polypeptide B. The polypeptide A and the polypeptide B prepared by the preparation method have the effects of inhibiting the formation of nitrosamine and activating the activity of ADH respectively, the prepared product can be used as a functional food for preventing and treating alcoholism, protecting liver and inhibiting the formation of nitrosamine; and the preparation method has the advantages of simple process, high polypeptide yield, significant functional activity of the prepared polypeptide and good taste.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of defatted krill processing, in particular to a krill active peptide, a preparation method thereof and a functional food. BACKGROUND

[0002] Antarctic krill contains rich nutrients such as protein, fat, mineral elements and chitin. Defatted krill powder is a by-product obtained after the dry krill is crushed and krill oil is extracted, and the crude protein content is 60-75%. At present, it is mainly used as a raw material for aquatic feed. For the extraction of Antarctic krill protein and polypeptide, the existing technology has reported the extraction of krill protein and polypeptide by alkali dissolution and acid precipitation, but mass production has not been seen.

[0003] Because the yield of polypeptide obtained by hydrolyzing krill with acid protease is low, the reported enzyme preparations for preparing krill polypeptide are mainly alkaline protease (including trypsin), neutral protease and sulfhydryl protease. Although some patents or documents report the preparation of polypeptide by hydrolyzing krill protein with pepsin, trypsin or alkaline protease is used for combined hydrolysis. For example:

[0004] In the paper "Response Surface Optimization of Antarctic Krill Powder Peptide Preparation Process and α-Glucosidase Inhibitory Activity Analysis" by Lang Meng et al., defatted Antarctic krill powder was used as raw material, and neutral protease, complex protease, alkaline protease, flavor protease and animal protease were selected for hydrolysis to prepare α-glucosidase inhibitory peptides, and their extraction effects were compared;

[0005] In the paper "Improvement of Antarctic Krill Peptide on Lipid Metabolism of Hyperuricemia Mice and Its Mechanism" by Wang Qinghui, Antarctic krill was hydrolyzed by adding 2% neutral protease, and the polypeptide solution with a molecular weight of 400-2000 Da had the effect of reducing hyperuricemia;

[0006] In the paper "Experimental Study on Anti-fatigue and Hypoxia Tolerance of Antarctic Krill Defatted Protein Peptide" by Xu Kai, Antarctic krill defatted protein was used as raw material, and papain was added for hydrolysis, and the obtained polypeptide had the effects of hypoxia tolerance and anti-fatigue;

[0007] A method for preparing active peptides from Antarctic krill powder is disclosed in Chinese patent CN109943615B, published on June 28, 2019. The scheme is as follows: Antarctic krill powder is first hydrolyzed with pepsin, then hydrolyzed with trypsin, and finally hydrolyzed three times with flavor protease to obtain Antarctic krill active peptides with the effect of scavenging free radicals;

[0008] In the Chinese patent application with the application number CN202210115111.1 and the publication date of 20220517, a kind of marine Antarctic krill peptide is disclosed, and the scheme is as follows: first, add trypsin to krill powder and hydrolyze for 7 hours, then add pepsin and hydrolyze for another 7 hours, and finally add papain and hydrolyze for 6 hours. The polypeptide hydrolysate obtained has the best effect on reducing cholesterol.

[0009] It is well known to those skilled in the art that alcohol dehydrogenase is the main pathway for liver metabolism of alcohol, and has the effect of alcoholism prevention and liver protection. Nitrosamine compounds are a kind of chemical carcinogens, and long-term low-dose exposure can cause cancer. Nitrite in meat products can be easily converted into nitrosamine under the action of gastrointestinal microorganisms. Therefore, substances that inhibit the formation of nitrosamine and activate the activity of alcohol dehydrogenase (ADH) are beneficial to human health when applied to the human body.

[0010] In the prior art, there is no report on the preparation of active polypeptides that can inhibit the formation of nitrosamine by enzymatic hydrolysis of defatted krill powder. There is also no report on the treatment of the residue after hydrolysis of krill by acid protease to obtain active peptides that can activate alcohol dehydrogenase (ADH). There is no report on the preparation method of polypeptides with nitrosamine formation inhibition activity and polypeptides with alcohol dehydrogenase (ADH) activity using the same defatted krill powder. SUMMARY

[0011] To solve the problems of the prior art mentioned in the background, the present application provides a preparation method of krill active peptides, which can use the same defatted krill powder to prepare polypeptides that inhibit the synthesis of nitrosamine and polypeptides that activate the activity of alcohol dehydrogenase (ADH). The technical scheme of the preparation method of krill active peptides is as follows:

[0012] The preparation method of krill active peptides comprises the following steps:

[0013] Defatted krill powder is mixed with water to form a first liquid; Aspergillus oryzae acid protease and pepsin are added to the first liquid for the first hydrolysis, and the enzyme is inactivated and separated to obtain supernatant A and precipitate B;

[0014] The supernatant A is filtered by membrane to obtain polypeptide A with a molecular weight of 200-1000 Da; polypeptide A has the effect of inhibiting the formation of nitrosamine;

[0015] The precipitate B is mixed with water to form a second liquid; ficin is added to the second liquid for the second hydrolysis, and the enzyme is inactivated and separated to obtain supernatant B;

[0016] The supernatant B is filtered by membrane to obtain polypeptide B with a molecular weight of less than 3000 Da, and polypeptide B has the effect of activating ADH activity.

[0017] In one embodiment, it includes the following steps:

[0018] The defatted krill powder is mixed with water at 80-90°C at a mass ratio of 1:(10-15) to obtain the first liquid mixture;

[0019] The pH of the first liquid was adjusted to 3.5-4.6. 2000-4000 U / g of Aspergillus oryzae acidic protease and 800-1500 U / g of pepsin were added according to the protein content in the defatted krill powder to carry out the first hydrolysis to obtain the first hydrolysate. The enzyme was inactivated and centrifuged to obtain supernatant A and precipitate B.

[0020] The supernatant A is filtered through a membrane to retain polypeptide A with a molecular weight cutoff of 200–1000 Da.

[0021] The precipitate B is mixed with water to form a second solution; wherein the mass ratio of the defatted krill powder to the water is 1:(8-10).

[0022] The pH of the second liquid was adjusted to 7.0-8.5. After high-temperature treatment, 2000-4000 U / g of fig protease was added according to the protein content in the defatted krill powder for a second hydrolysis to obtain the second hydrolysate. The enzyme was inactivated and centrifuged to obtain the supernatant B.

[0023] The supernatant B is filtered through a membrane to remove polypeptide B with a molecular weight cutoff of less than 3000 Da.

[0024] In one embodiment, the temperature of the first hydrolysis is 35-45°C and the time is 2.5-4 hours; the temperature of the second hydrolysis is 50-65°C and the time is 90-150 minutes.

[0025] In one embodiment, the first hydrolysate is subjected to enzyme inactivation treatment at 75-85°C for 6-10 minutes; the second solution is subjected to high-temperature treatment: the second solution is placed in a high-pressure steam cooker and heated to 120-133°C, and maintained for 10-20 minutes; the second hydrolysate is subjected to enzyme inactivation treatment at 98-100°C for 10-15 minutes.

[0026] In one embodiment, the supernatant A is filtered through a membrane to obtain a retentate containing polypeptide A with a molecular weight of 200-1000 Da. The retentate is then concentrated and dried to obtain polypeptide A powder. The supernatant B is filtered through a membrane to obtain an ultrafiltrate containing polypeptide B with a molecular weight of less than 3000 Da. The ultrafiltrate is then concentrated and dried to obtain polypeptide B powder.

[0027] In one embodiment, the supernatant A is sequentially subjected to microfiltration and ultrafiltration through a 1000 Da ultrafiltration membrane to obtain a filtrate. The filtrate is then subjected to nanofiltration through a 200 Da nanofiltration membrane to obtain a retentate containing polypeptide A with a molecular weight of 200-1000 Da. The second hydrolysate is centrifuged at 7000-10000 rpm for 8-15 min to obtain supernatant B. Supernatant B is subjected to ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltrate containing polypeptide B with a molecular weight of less than 3000 Da.

[0028] In one embodiment, the defatted krill powder is mixed with water, the mixture is ground into a slurry using a colloid mill, and the sieve is filtered through a 100-150 mesh screen to obtain the sieve-through material, thus obtaining the first liquid.

[0029] In one embodiment, when polypeptide A is dried to form polypeptide A powder and diluted to a concentration of 0.6–2.6 mg / ml, its inhibition rate against nitrosamine formation is 56–93%; when polypeptide B is dried to form polypeptide B powder and diluted to a concentration of 10–40 mg / ml, its activation rate against alcohol dehydrogenase is above 44%.

[0030] The present invention also provides a krill active peptide, which includes polypeptide A and / or polypeptide B; wherein the krill active peptide is prepared by the krill active peptide preparation method described above.

[0031] Based on the above, compared with the prior art, the method for preparing krill active peptides provided by the present invention has the following beneficial effects:

[0032] This method can prepare two peptides using the same defatted krill powder: one with activity to inhibit nitrosamine formation and the other with activity to activate alcohol dehydrogenase (ADH). Furthermore, this preparation method has the following advantages:

[0033] (1) Simple process: Existing methods for extracting krill polypeptides mostly use alkali dissolution and acid precipitation to extract krill protein, and then use protease to hydrolyze it; This invention directly adds enzyme to defatted krill powder for hydrolysis, without the alkali dissolution and acid precipitation process for protein extraction, making the process simpler and reducing chemical wastewater discharge.

[0034] (2) High polypeptide yield: The preparation method of this invention uses three enzymes for hydrolysis, which results in high yield of the two polypeptides and good utilization of krill protein;

[0035] (3) The obtained polypeptides have significant functional activity: The two polypeptides obtained by the present invention have the functions of inhibiting the formation of nitrosamines and activating ADH, respectively. Therefore, the two polypeptides obtained by the present invention can be used as raw materials for functional products that maintain human health, such as relieving hangovers, protecting the liver, and inhibiting the formation of nitrosamines.

[0036] (4) The obtained polypeptides have a good taste: Commonly used neutral protease, alkaline protease or papain hydrolyze krill liquid and have a bitter taste. However, in the first hydrolysis, Aspergillus oryzae acid protease and pepsin are added to defatted krill in a certain proportion for hydrolysis. The resulting polypeptide A has no bitter taste and polypeptide B has a slight bitter taste. The good taste is conducive to improving the user experience.

[0037] 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

[0038] 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.

[0039] Figure 1 The blocking rates of ascorbic acid and polypeptides of different molecular weight ranges against nitrosamines at different concentrations in Example 3 of this invention;

[0040] Figure 2 The activation rates of ADH by polypeptide B of different molecular weight ranges and unfiltered crude peptide powder at different concentrations in Example 3 of the present invention. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] This invention also provides an operational example of a method for preparing krill bioactive peptides, the specific steps of which are as follows:

[0044] (1) Add 10 to 15 times the weight of hot water at 80 to 90°C to defatted krill powder, grind it into a slurry using a colloid mill, and filter it through a 100 to 150 mesh screen. Take the sieve material to obtain the first liquid.

[0045] (2) Adjust the pH of the feed solution to 3.5–4.6, and add 2000–4000 U / g of Aspergillus oryzae acidic protease and 800–1500 U / g of pepsin according to the protein content in the raw material for the first hydrolysis. The hydrolysis temperature is 35–45℃ and the time is 2.5–4 h. After the first hydrolysis, inactivate the enzymes at 75–85℃ for 6–10 min, and then obtain the supernatant A and precipitate B by centrifugation. The temperature of the first hydrolysis is 35–45℃ and the time is 2.5–4 h.

[0046] (3) The supernatant A is subjected to microfiltration and ultrafiltration with a 1000 Da ultrafiltration membrane to obtain filtrate. The filtrate is then subjected to nanofiltration with a 200 Da nanofiltration membrane to obtain a retentate containing polypeptide A with a molecular weight of 200-1000 Da. The retentate is concentrated and dried to obtain polypeptide A powder with the function of inhibiting the formation of nitrosamines.

[0047] (4) Add 8 to 10 times the weight of the krill powder in water to the precipitate B, adjust the pH to 7.0 to 8.5, stir well, put it into a high-pressure steam cooker, heat to 120 to 133°C, and keep for 10 to 20 minutes.

[0048] (5) Then, add 2000-4000 U / g of fig protease according to the amount of protein in the raw material for a second hydrolysis. The hydrolysis temperature is 50-65℃ and the time is 90-150 min. After hydrolysis, inactivate the enzyme at 98-100℃ for 10-15 min, and then centrifuge at 7000-10000 rpm for 8-15 min to obtain supernatant B.

[0049] (6) The supernatant B is subjected to ultrafiltration treatment with a 3000 Da ultrafiltration membrane to obtain an ultrafiltrate containing polypeptide B with a molecular weight of less than 3000 Da. The ultrafiltrate is concentrated and dried to obtain polypeptide B powder.

[0050] Among them, the prepared polypeptide A has the effect of inhibiting nitrosamine synthesis. When the polypeptide A powder prepared using the method of this application is diluted to a concentration of 0.6-2.6 mg / ml, the inhibition rate of nitrosamine formation is 56-93%. The prepared polypeptide B has the effect of activating alcohol dehydrogenase activity. When the polypeptide B powder prepared using the method of this application is diluted to a concentration of 10-40 mg / ml, the activation rate of alcohol dehydrogenase is more than 44%.

[0051] The two polypeptides obtained using this invention possess the functions of inhibiting nitrosamine formation and activating ADH, respectively. Alcohol dehydrogenase (ADH) is the main pathway for alcohol metabolism in the liver, thus playing a role in detoxification and liver protection. Nitrosamines are chemical carcinogens; long-term, low-dose exposure can cause cancer. Nitrites in meat products are easily converted into nitrosamines by gastrointestinal microorganisms. Therefore, the two polypeptides obtained in this invention can serve as functional raw materials for maintaining human health through detoxification, liver protection, and inhibition of nitrosamine formation.

[0052] It should be noted that:

[0053] The krill active peptides of this application can be used as raw materials for functional products, but their effects include, but are not limited to, hangover relief and liver protection. Other health benefits they can provide by inhibiting the formation of nitrosamines and activating ADH function in the human body are also applicable. Similarly, based on the polypeptides obtained in this application, which have the functions of inhibiting nitrosamine formation and activating ADH, polypeptides A and B can also be used in other forms of products, such as pharmaceuticals, health products, etc., including any substance products that provide preventative and / or other beneficial effects.

[0054] To verify the effectiveness of the proposed solution, this application also provides the following embodiments and comparative examples:

[0055] Example 1

[0056] The preparation process of this embodiment is as follows:

[0057] 1. Raw material pretreatment: 100g of finely crushed defatted krill powder (protein content of 69.5%) is added to 1500g of purified water at 90℃. After grinding the mixture into a slurry using a colloid mill, the liquid is filtered through a 120-mesh sieve.

[0058] 2. First hydrolysis and preparation of peptide A powder: The pH of the solution was adjusted to 3.5, and 270,000 U of Aspergillus oryzae acidic protease (Amano Amano MSD protease) and 60,000 U of pepsin were added. Hydrolysis was carried out at 36℃ for 4 hours. After hydrolysis, the enzyme was inactivated at 85℃ for 6 minutes. The enzyme-inactivated solution was centrifuged to obtain supernatant A and precipitate B. Supernatant A was first microfiltered, and the filtrate was ultrafiltered using a 1000 Da ultrafiltration membrane. The filtrate was then nanofiltered and concentrated using a 200 Da nanofiltration membrane. The resulting concentrated retentate A was spray-dried to obtain peptide A powder with the function of inhibiting the formation of nitrosamines.

[0059] The test showed that when peptide A powder was diluted to 1.2 mg / ml, the blocking rate against nitrosamines was 83.5%.

[0060] 3. Second hydrolysis and preparation of peptide B powder: Precipitate B was added to 800 g of water, the pH was adjusted to 8.3, and the mixture was stirred. It was then placed in an autoclave and heated to 121°C for 15 min. After cooling, 150,000 U of fig protease was added, and hydrolysis was carried out at 65°C for 90 min. After two hydrolyses, the enzyme was inactivated at 98°C for 15 min, followed by centrifugation at 8000 rpm for 12 min. The supernatant was ultrafiltered using a membrane with a molecular weight cutoff of 3000 Da. The filtrate B was concentrated under vacuum and spray-dried to obtain peptide B powder with ADH-activating activity. At a peptide concentration of 20 mg / ml, the peptide B powder showed an ADH activation rate of 65.9%.

[0061] Comparative Example 1

[0062] During the first hydrolysis, only an equal amount of Aspergillus oryzae acidic protease was added, without adding pepsin. The remaining processes and steps were the same as in Example 1.

[0063] Comparative Example 2

[0064] During the first hydrolysis, only an equal amount of pepsin was added, and Aspergillus oryzae acid protease was not added. The remaining processes and steps were the same as in Example 1.

[0065] Comparative Example 3

[0066] During the first hydrolysis, Aspergillus niger acidic protease was replaced with an equal amount of Aspergillus oryzae acidic protease, and the remaining processes and steps were the same as in Example 1.

[0067] Comparative Example 4

[0068] During the first hydrolysis, Bacillus licheniformis acidic protease was used to replace Aspergillus oryzae acidic protease with an equal amount of enzyme, and the remaining processes and steps were the same as in Example 1.

[0069] Comparative Example 5

[0070] During the first hydrolysis, pepsin was replaced with an equal amount of Bacillus licheniformis acidic protease, and the remaining processes and steps were the same as in Example 1.

[0071] Comparative Example 6

[0072] During the first hydrolysis, pepsin was replaced with an equal amount of Aspergillus oryzae acidic protease, and the remaining processes and steps were the same as in Example 1.

[0073] The test results of Example 1 and Comparative Examples 1-6 are shown in Table 1:

[0074] Table 1

[0075]

[0076] In Table 1, "-" indicates no inhibition or no activation.

[0077] The detection data from Example 1 and Comparative Examples 1-6 show that:

[0078] (1) In the first enzymatic hydrolysis, the nitrosamine inhibition rate of the defatting krill powder with Aspergillus oryzae acidic protease was certain. However, only Example 1, which was hydrolyzed by a combination of Aspergillus oryzae acidic protease and pepsin, had a high nitrosamine inhibition rate. All other enzymatic hydrolysis methods that replaced Aspergillus oryzae acidic protease or pepsin with the same amount of enzyme could not produce a high nitrosamine inhibition rate and alcohol dehydrogenase activation rate.

[0079] (2) Regarding the total polypeptide yield of polypeptide A and polypeptide B: the total polypeptide yield of Comparative Examples 3, 4, 5, and 6 was all above 50%. However, Example 1 not only had a total polypeptide yield exceeding 60%, but also had a good taste with no bitterness. Comparative Examples 3 and 4 showed neither nitrosamine inhibitory activity nor ADH activating activity; although Comparative Examples 5 and 6 showed nitrosamine inhibitory activity, it was far lower than that of Example 1, and the polypeptide A liquid tasted bitter. Comparative Example 5 showed ADH activating activity, but the activity was very low.

[0080] Example 2

[0081] The preparation process of this embodiment is as follows:

[0082] 1. Raw material pretreatment: 600g of finely crushed defatted krill powder (protein content 69.5%) is added to 6000g of purified water at 80℃. After grinding the mixture into a slurry using a colloid mill, the liquid is filtered through a 150-mesh sieve.

[0083] 2. First hydrolysis and preparation of peptide A powder: The pH of the feed solution was adjusted to 4.5, and 950,000 U of Aspergillus oryzae acidic protease and 600,000 U of pepsin were added. Hydrolysis was carried out at 40℃ for 3.5 h. After hydrolysis, the enzymes were inactivated at 80℃ for 8 min. The enzyme-inactivated solution was centrifuged to obtain supernatant A and precipitate B. Supernatant A was first microfiltered, and the filtrate was ultrafiltered using a 1000 Da ultrafiltration membrane. The filtrate was then nanofiltered and concentrated using a 200 Da nanofiltration membrane. The resulting concentrated retentate A was freeze-dried to obtain peptide A powder, which has the effect of inhibiting the formation of nitrosamines. When peptide A powder was diluted to 1.8 mg / ml, the inhibition rate of nitrosamine formation was 94.10%; the peptide yield relative to krill protein was 21.45%.

[0084] 3. Second hydrolysis and preparation of peptide B powder: Take 1 / 6 of the total weight of precipitate B, add 1000 g of water, adjust the pH to 7.0, stir well, and place in a high-pressure steam cooker. Heat to 126℃ and maintain for 20 min. Then add 160,000 U of fig protease and hydrolyze at 60℃ for 120 min. After two hydrolyses, inactivate the enzyme at 100℃ for 10 min, then centrifuge at 10,000 rpm for 8 min. Filter the supernatant through a 3000 Da ultrafiltration membrane. The filtrate B is concentrated under vacuum and freeze-dried to obtain peptide B powder with ADH activation. Note: In the second hydrolysis, since the amount of precipitate B in this step is one-sixth of the total amount, the amount of water and fig protease added is converted to one-sixth of the weight of defatted krill powder.

[0085] The activation rate of ADH by peptide B powder at a concentration of 30 mg / ml was measured to be 64.37%, and the peptide yield was 37.63%.

[0086] Comparative Example 7

[0087] During the second hydrolysis, the fig protease was replaced with an equal amount of thermophilic lipolytic Bacillus protease, and the hydrolysis temperature was 65°C; the remaining process conditions and steps were the same as in Example 2.

[0088] Comparative Example 8

[0089] During the second hydrolysis, the fig protease was replaced with an equal amount of Bacillus amyloliquefaciens neutral protease (Novozymes 0.8L protease), and the hydrolysis temperature was 50°C; the remaining process conditions and steps were the same as in Example 2.

[0090] Comparative Example 9

[0091] During the second hydrolysis, the fig protease was replaced with an equal amount of Bacillus subtilis neutral protease, and the hydrolysis temperature was 55°C; the remaining process conditions and steps were the same as in Example 2.

[0092] Comparative Example 10

[0093] During the second hydrolysis, papain was used to replace fig protease in the same amount, and the hydrolysis pH was 6.5; the remaining process conditions and steps were the same as in Example 2.

[0094] Comparative Example 11

[0095] During the second hydrolysis, the fig protease was replaced with an equal amount of Bacillus licheniformis alkaline protease, the hydrolysis temperature was 50°C, and the hydrolysis pH was 8.5; the remaining process conditions and steps were the same as in Example 2.

[0096] This invention also includes several comparative examples, the results of which show that the hydrolysis effect is not as good as that of Example 2, but they are not listed here due to space limitations.

[0097] The test results of the products of Example 2 and Comparative Examples 7-11 are shown in Table 2:

[0098] Table 2

[0099]

[0100] In Table 2, the "-" indicates no inhibition or no activation.

[0101] The detection data from Example 2 and Comparative Examples 7-11 show that:

[0102] (1) During the second enzymatic hydrolysis, the fig protease was replaced with neutral and alkaline protease and papain from different strains with the same amount of enzyme. Since the enzymes used in the above replacements are suitable for different acid-base environments (or pH environments), the pH value of the solution was also adjusted in a synchronous manner to adapt to the use of different enzymes. The results showed that all hydrolysates of Comparative Examples 7-11 had no ADH activation activity.

[0103] (2) In terms of peptide yield and taste, except for Comparative Examples 9 and 11, whose peptide yields were slightly higher than those of Example 2, the others were lower than those of Example 2; however, Comparative Examples 9 and 11 had a strong bitter taste and poor taste. In this case, only Example 2 not only had a high ADH activation rate but also a relatively high peptide yield and a mild bitter taste, making it an ideal process choice.

[0104] Example 3

[0105] The preparation process of this embodiment is as follows:

[0106] 1. Raw material pretreatment: 100g of finely crushed defatted krill powder (protein content 69.5%) is added to 1200g of purified water at 85℃. After grinding the mixture into a slurry using a colloid mill, the liquid is filtered through a 120-mesh sieve.

[0107] 2. First hydrolysis and preparation of polypeptide A powder: Adjust the pH of the solution to 4.0, add 210,000 U of Aspergillus oryzae acidic protease and 70,000 U of pepsin, and hydrolyze at 45℃ for 2.5 h. After hydrolysis, inactivate the enzymes at 75℃ for 10 min; centrifuge the enzyme-inactivated solution to obtain supernatant A and precipitate B.

[0108] After microfiltration of supernatant A, it was separated using ultrafiltration membranes with molecular weight cutoffs of 3000 Da and 1000 Da, and a nanofiltration membrane with a molecular weight cutoff of 200 Da, yielding four components: molecular weight Mw > 3000 Da (A1), Mw between 1000 and 3000 Da (A2), Mw between 200 and 1000 Da (A3), and microfiltrate (A0). Each of the four components was vacuum concentrated and freeze-dried into powder. The peptide powders of three different molecular weight segments and one microfiltration crude peptide powder were diluted with distilled water to specific concentrations. The blocking rates of peptides of different molecular weight segments against nitrosamines at different concentrations were measured, and compared with ascorbic acid. Specific results are shown in [link to results]. Figure 1 .

[0109] 3. Second hydrolysis and preparation of peptide B powder: Take precipitate B and add 1000g of water, adjust the pH to 8.0, stir well and put it into a high-pressure steam cooker, heat to 126℃ and keep for 15min; then add 220,000U of fig protease and hydrolyze at 50℃ for 150min.

[0110] After two hydrolysis cycles, the enzyme was inactivated at 100℃ for 10 min, followed by centrifugation at 10000 rpm for 10 min. The supernatant was separated using ultrafiltration membranes with molecular weight cutoffs of 10000 Da, 3000 Da, and 1000 Da to obtain five fractions: Mw>3000–10000 Da (B1), Mw1000–3000 Da (B2), Mw<3000 Da (B3), Mw<1000 Da (B4), and unfiltered centrifuged liquid (B0). Each fraction was vacuum concentrated and freeze-dried into powder. The peptide powders (peptides B of different molecular weight ranges) and the unfiltered crude peptide powder were diluted with distilled water to specific concentrations, and the ADH activation rate of each fraction was measured. The results of the activation rates at different concentrations are shown in [link to data]. Figure 2 .

[0111] The detection data from Example 3 show that:

[0112] (1) Analysis Figure 1 It can be seen that among the four components of polypeptide A, A1 (>3000Da), A2 (1000~3000Da), A3 (200~1000Da) and A0 (unfiltered microfiltrate), component A3 has the best blocking effect on nitrosamine synthesis. At a concentration of 0.6~2.6 mg / ml, this segment of polypeptide A has an inhibition rate of 56~93% against nitrosamines, which is better than that of ascorbic acid.

[0113] However, experimental results showed that when the concentration of peptide A exceeded 2.6 mg / ml, the inhibition rate of nitrosamines decreased rapidly. Figure 1 (This phenomenon was not shown in the text; the results are described here.) The analysis suggests that the interference effect of inactive peptides or hydrolyzed chitosan oligosaccharides in the multi-peptide solution increases with increasing concentration. Furthermore, previous experiments also found that if small molecules are not removed by nanofiltration, the inhibition rate of nitrosamines decreases more rapidly in component A3. Figure 1 (This phenomenon is not shown for the unfiltered A3 fraction; the results are described in words here.) Although the A2 fraction also exhibits inhibitory activity, it is far lower than that of the A3 fraction, especially when the concentration of peptide A is higher than 1.4 mg / ml. Therefore, the 200–1000 Da ultrafiltration fraction of peptide A was selected as the optimal peptide for inhibiting nitrosamine synthesis.

[0114] (2) Through Figure 2 Analysis shows that:

[0115] Among the five components of peptide B, the ADH activation rate was very low in the 3000–10000 Da (B1) range, while the 1000–3000 Da (B2), <3000 Da (B3), <1000 Da (B4) range and the unfiltered centrifuged liquid (B0) all showed some ADH activation activity. This indicates that ADH-activating peptides exist in both the 1000–3000 Da and <1000 Da ultrafiltration ranges, with the highest ADH activation activity observed in the <3000 Da range. In this range, the ADH activation rate was 44–68% at a concentration of 10–40 mg / ml.

[0116] However, the ADH activation rate of peptide B decreased in all ultrafiltration segments after the peptide concentration exceeded a certain range. This may be because the ultrafiltration solution is a mixture of various peptides, and also contains minerals and water-soluble chitosan. When the peptide concentration exceeds a certain range, the interference of other impurities on the ADH-activating peptide becomes increasingly significant. Therefore, when using peptide B as the ADH-activating peptide, the ultrafiltration segment with peptide B < 3000 Da is selected as the optimal peptide segment, and the preferred peptide B concentration is controlled between 10 and 40 mg / ml.

[0117] In summary, by analyzing the inhibition rate of peptide A against nitrosamines at different concentrations and the ADH activation rate of peptide B at different concentrations, it can be seen that the preferred concentration of peptide A is controlled at 0.6–2.6 mg / ml, and the preferred concentration of peptide B is controlled at 10–40 mg / ml. This preferred concentration range can guide the usage of peptide A and peptide B during the application stage. For example, when peptide A powder or peptide B powder is prepared into a solution for oral administration, the peptide A solution and peptide B solution can be prepared and used according to the above preferred concentration range in order to obtain better results.

[0118] The results characterization test methods for the examples and comparative examples are as follows:

[0119] (1) The method for determining and calculating the nitrosamine inhibition rate of polypeptide A is as follows:

[0120] Accurately pipette different volumes of peptide samples into 10 mL volumetric flasks, and sequentially add 5 mL of pH 3.0 citrate-disodium hydrogen phosphate buffer, 0.5 mL of 1 mmol / L NaNO₂ solution, and 0.5 mL of 25 mmol / L diethylamine. Dilute to the mark and incubate at 37°C for 2 hours. Pipe 1.0 mL of the above reaction solution into a 7 cm⁻¹ volumetric flask. 2 Add 0.5 mL of 0.5% Na2CO3 solution to the culture dish, place it on a UV analyzer covered with black cloth and irradiate for 1 h with the UV lamp 15 cm away from the liquid surface. After removing it, add 1.5 mL each of 1% (mass fraction) p-aminobenzenesulfonic acid and 0.1% (mass fraction) α-naphthylamine solution, add water to make the exact volume of the solution 5.0 mL, shake well and let stand for 45 min, then measure the absorbance value at 525 nm and calculate the inhibition rate.

[0121] Where, inhibition rate = (A0~A X ) / A0×100%. Where A x A0 refers to the absorbance value after adding the sample, while A0 refers to the absorbance value without adding the sample.

[0122] (2) Test method for ADH activation rate:

[0123] ADH activity was determined using an ADH detection kit. The formula for calculating the ADH activation rate A (%) is: A = (E 样品 —E 空白 ) / E 空白 ×100. Where: E 样品 —Enzyme activity (U) in polypeptide sample reaction solution; E 空白 Enzyme activity (U) of a peptide-free blank reaction solution.

[0124] (3) The polypeptide yield is calculated as follows:

[0125] Peptide yield % = (peptide concentration in hydrolysate × hydrolysate volume × 100%) / amount of hydrolyzed protein.

[0126] (4) Taste of polypeptide solution: Prepare a 10 mg / ml solution of polypeptide powder using distilled water as the diluent. Five trained tasters will taste and score the solution, with four levels from best to worst: no bitterness, slightly bitter, very bitter, and very bitter. The specific scoring criteria for the four levels are shown in Table 3 below. After tasting, the scores of the five tasters will be added together. A total score of 4 or less indicates no bitterness; 5-24 indicates slightly bitter; 25-50 indicates very bitter; and 51 or more indicates very bitter.

[0127] Table 3

[0128]

[0129] In summary, the method for preparing krill bioactive peptides provided by this invention has at least the following effects and technical benefits:

[0130] This method can prepare two peptides using the same defatted krill powder: one with activity to inhibit nitrosamine formation and the other with activity to activate alcohol dehydrogenase (ADH). Furthermore, this preparation method has the following advantages:

[0131] (1) Simple process: Existing methods for extracting krill polypeptides mostly use alkali dissolution and acid precipitation to extract krill protein, and then use protease to hydrolyze it; This invention directly adds enzyme to defatted krill powder for hydrolysis, without the alkali dissolution and acid precipitation process for protein extraction, making the process simpler and reducing chemical wastewater discharge.

[0132] (2) High peptide yield: The preparation method of the present invention involves two hydrolysis processes of defatted krill powder by acid and neutral protease, combined with fine grinding and high-temperature treatment of the raw material slurry, resulting in a peptide yield of 54-60%. The two peptides obtained have high yields and good utilization of krill protein.

[0133] (3) The obtained polypeptides exhibit significant functional activity: The two polypeptides obtained using this invention respectively have the functions of inhibiting nitrosamine formation and activating ADH. Alcohol dehydrogenase is the main pathway for liver metabolism of alcohol and has the effect of detoxifying alcohol and protecting the liver. Nitrosamines are chemical carcinogens, and long-term low-dose exposure can cause cancer. Nitrites in meat products are easily converted into nitrosamines under the action of gastrointestinal microorganisms. Therefore, the two polypeptides obtained in this invention can be used as raw materials for functional products that maintain human health by detoxifying alcohol, protecting the liver, and inhibiting the formation of nitrosamines.

[0134] (4) The obtained polypeptides have a good taste: Commonly used neutral protease, alkaline protease and papain hydrolyze krill liquid all have a bitter taste. However, in the first hydrolysis, Aspergillus oryzae acidic protease and pepsin are added to defatted krill in a certain proportion for hydrolysis. The resulting polypeptide A has no bitter taste and polypeptide B has a slight bitter taste, which is good and helps to improve the user experience.

[0135] It should be noted that:

[0136] In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0137] The Aspergillus oryzae acidic protease, pepsin, and fig protease used in the embodiments and comparative examples of this application are all existing enzymes; the Aspergillus oryzae acidic protease used in the embodiments and comparative examples is MSD acidic protease produced by Amano Co., Ltd., but is not limited thereto.

[0138] The relevant terms in this article are defined and explained as follows:

[0139] "ADH" is the common abbreviation for "ethanol dehydrogenase" in this field, and the activation rate of "ADH" refers to the "activation rate of ethanol dehydrogenase".

[0140] The term "microfiltration" as used in this article, also known as microporous filtration, is a type of precision filtration. Microfiltration can trap particles between 0.1 and 1 micrometer. Microfiltration membranes allow large organic molecules and inorganic salts to pass through, but can block suspended solids, bacteria, some viruses, and large-scale colloids. "Microfiltration" is a commonly used term in this field, and its name accurately describes the process; therefore, it will not be elaborated upon here.

[0141] The enzyme activity unit "U" used in this article refers to the amount of enzyme that can convert 1 micromolar of substrate in 1 minute under specific conditions (25℃, other conditions being optimal), or the amount of enzyme that can convert 1 micromolar of a related group in the substrate. It is called one International Unit (IU, also known as U). Enzyme activity is also called enzyme activity.

[0142] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0143] 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.

[0144] 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.

[0145] 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 krill bioactive peptides, characterized in that, Includes the following steps: Defatted krill powder is mixed with water to form a first solution; the pH of the first solution is adjusted to 3.5-4.6, and 2000-4000 U / g of Aspergillus oryzae acidic protease and 800-1500 U / g of pepsin are added according to the protein content of the defatted krill powder to carry out the first hydrolysis, obtaining the first hydrolysate. The first hydrolysate is then subjected to enzyme inactivation treatment and centrifugation to obtain supernatant A and precipitate B; the temperature of the first hydrolysis is 35-45℃ and the time is 2.5-4 hours. The supernatant A is filtered through a membrane to retain polypeptide A with a molecular weight cutoff of 200–1000 Da. The precipitate B is mixed with water to form a second solution; the pH of the second solution is adjusted to 7.0-8.5, and after high-temperature treatment, 2000-4000 U / g of fig protease is added according to the protein content in the defatted krill powder for a second hydrolysis to obtain a second hydrolysate. The second hydrolysate is then inactivated and centrifuged to obtain supernatant B; the temperature of the second hydrolysis is 50-65℃, and the time is 90-150 min. The supernatant B is filtered through a membrane to remove polypeptide B with a molecular weight cutoff of less than 3000 Da.

2. The method for preparing krill bioactive peptides according to claim 1, characterized in that: It includes the following steps: The defatted krill powder is mixed with water at 80-90°C at a mass ratio of 1:(10-15) to obtain the first liquid mixture; The pH of the first liquid was adjusted to 3.5-4.

6. 2000-4000 U / g of Aspergillus oryzae acidic protease and 800-1500 U / g of pepsin were added according to the protein content in the defatted krill powder to carry out the first hydrolysis to obtain the first hydrolysate. The enzyme was inactivated and centrifuged to obtain supernatant A and precipitate B. The supernatant A is filtered through a membrane to retain polypeptide A with a molecular weight cutoff of 200–1000 Da. The precipitate B is mixed with water to form a second solution; wherein the mass ratio of the defatted krill powder to the water is 1:(8-10). The pH of the second liquid was adjusted to 7.0-8.

5. After high-temperature treatment, 2000-4000 U / g of fig protease was added according to the protein content in the defatted krill powder for a second hydrolysis to obtain the second hydrolysate. The enzyme was inactivated and centrifuged to obtain the supernatant B. The supernatant B is filtered through a membrane to remove polypeptide B with a molecular weight cutoff of less than 3000 Da.

3. The method for preparing krill bioactive peptides according to any one of claims 1 to 2, characterized in that: The first hydrolysis was performed at a temperature of 35–45°C for 2.5–4 hours. The second hydrolysis is performed at a temperature of 50–65°C for a time of 90–150 min.

4. The method for preparing krill bioactive peptides according to any one of claims 1 to 2, characterized in that: The first hydrolysate was subjected to enzyme inactivation treatment at 75–85°C for 6–10 min; The second liquid is subjected to high-temperature treatment: the second liquid is placed in a high-pressure steam cooker and heated to 120-133°C for 10-20 minutes; the second hydrolysate is treated with enzyme inactivation at 98-100°C for 10-15 minutes.

5. The method for preparing krill bioactive peptides according to any one of claims 1 to 2, characterized in that: The supernatant A is filtered through a membrane to obtain a retentate containing polypeptide A with a molecular weight of 200-1000 Da. The retentate is then concentrated and dried to obtain polypeptide A powder. The supernatant B is filtered through a membrane to obtain an ultrafiltrate containing polypeptide B with a molecular weight of less than 3000 Da. The ultrafiltrate is then concentrated and dried to obtain polypeptide B powder.

6. The method for preparing krill bioactive peptides according to claim 5, characterized in that: The supernatant A is subjected to microfiltration and ultrafiltration through a 1000 Da ultrafiltration membrane to obtain a filtrate. The filtrate is then subjected to nanofiltration through a 200 Da nanofiltration membrane to obtain a retentate containing polypeptide A with a molecular weight of 200-1000 Da. The second hydrolysate is centrifuged at 7000-10000 rpm for 8-15 min to obtain supernatant B; the supernatant B is then subjected to ultrafiltration through a 3000 Da ultrafiltration membrane to obtain an ultrafiltrate containing polypeptide B with a molecular weight of less than 3000 Da.

7. The method for preparing krill bioactive peptides according to any one of claims 1 to 2, characterized in that: The defatted krill powder is mixed with water, the mixture is ground into a slurry using a colloid mill, and the sieve is filtered through a 100-150 mesh screen to obtain the sieve material, thus obtaining the first liquid.

8. The method for preparing krill bioactive peptides according to any one of claims 1 to 2, characterized in that: When the polypeptide A is dried to form polypeptide A powder and diluted to a concentration of 0.6–2.6 mg / ml, its inhibition rate against nitrosamine formation is 56–93%. The polypeptide B is dried to form polypeptide B powder, and when diluted to a concentration of 10-40 mg / ml, its activation rate for alcohol dehydrogenase is over 44%.

9. Krill bioactive peptides, characterized in that: It includes polypeptide A and / or polypeptide B; The krill active peptide is prepared by the method for preparing krill active peptide as described in any one of claims 1 to 8.

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