A method for preparing skipjack tuna bioactive peptides and a composition thereof

Abstract

This invention relates to the field of deep processing technology for marine fish foods, and particularly to a method and composition for preparing skipjack tuna active peptides. The preparation method includes the following steps: mixing skipjack tuna powder with water, keeping it warm and allowing it to swell, and then grinding it into a slurry using a colloid mill; adding Aspergillus protease to the slurry for hydrolysis to obtain a hydrolysate; inactivating the enzyme in the hydrolysate, centrifuging to remove impurities, and obtaining a supernatant; separating the supernatant through a membrane to retain the peptides, thus obtaining the skipjack tuna active peptides. This invention provides skipjack tuna active peptides with the property of protecting pepsin activity, which can be used as a pepsin protectant in the food, agriculture, animal husbandry, or feed processing industries, and can also be used as a raw material for sports nutrition foods.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology for marine fish food, and in particular to a method and composition for preparing bonito bioactive peptides. Background Technology

[0002] Pepsin is a digestive protease secreted by the chief cells of the gastric mucosa in the stomach. Its function is to break down proteins in food into small peptide fragments, and it is commonly used in the pharmaceutical, food processing, and agricultural product processing industries. Although it has excellent protein degradation capabilities, it easily loses its activity at temperatures above 60°C or at pH values ​​greater than 5, which limits its applications. Therefore, improving the activity and stability of pepsin has always been a problem that needs to be solved in the industry.

[0003] Current research on improving pepsin stability includes: Bian Jinlin's report in "The Promoting Effect of Ya'an Tibetan Tea on Pepsin" states that catechins in Ya'an Tibetan tea can enhance pepsin activity; the cited references in this article report that acidic substances such as organic acids, amino acids, and vitamin C can effectively enhance protease activity. Liu Xinze's research in "The Effect of Adding Different Combinations of Acidifying Agents to Diets on Pepsin Activity" suggests that adding 0.1% (ammonium formate + essential oil) compound acidifying agent to the diet increased pepsin activity in broilers.

[0004] Skipjack tuna is a low-fat, high-protein fish. Dried skipjack tuna meat meal contains over 80% protein and less than 5% fat. However, due to its strong fishy smell and poor taste, it is generally processed into low-value products such as dried fish and canned goods, and has not been further processed or utilized. Research on processing functional peptides from skipjack tuna includes: Qu Yushan's dissertation, "Preparation and Anti-fatigue Efficacy Study of Antioxidant Peptides from Skipjack Tuna," which used papain to enzymatically hydrolyze skipjack tuna to prepare oligopeptides with antioxidant and anti-fatigue effects. Li Yujuan, in her article "Preparation, Isolation, Structural Characterization and Efficacy Mechanism Study of Skipjack Tuna Uric Acid-Lowering Peptides," used papain to hydrolyze skipjack tuna protein to obtain oligopeptides with less than 1000 Da that inhibit xanthine oxidase (XO) activity in vitro. Huang Cheng, in his study "Physiological Activity Study of High-F-Value Oligopeptide Liquid from Skipjack Tuna Meat," used pepsin and flavor enzymes for stepwise hydrolysis to study the hangover-relieving and anti-fatigue effects of high-F-value oligopeptides. Chinese invention patent document CN116098984A discloses the application and experiment of using skipjack tuna oligopeptides in the process of liver damage repair.

[0005] In summary, no reports have yet been found of peptides that can protect pepsin or improve its thermal stability. 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 skipjack tuna bioactive peptides, comprising the following steps:

[0007] Mix bonito powder with water, keep it warm and allow it to swell, then grind it into a slurry using a colloid mill;

[0008] Honey Aspergillus protease was added to the slurry for hydrolysis to obtain the hydrolysate;

[0009] After inactivating the enzymes in the hydrolysate and removing impurities by centrifugation, the supernatant is obtained. The supernatant is then separated by membrane separation to retain the polypeptide, thus obtaining the bonito bioactive peptide.

[0010] The mass ratio of bonito meal to water is 1:(20-30);

[0011] The temperature for heat preservation and expansion is 60-70℃, and the time is 40-60 minutes;

[0012] The slurry obtained from the grinding process needs to be sieved through a 100-mesh filter screen;

[0013] The amount of honey aspergillin added is based on the protein content in the bonito powder, with an enzyme activity of 1000u-2000u per gram.

[0014] The pH of the slurry is adjusted to 7.5-8.5, the hydrolysis temperature is 38-48℃, and the hydrolysis time is 90-150 min;

[0015] First, adjust the pH of the hydrolysate to 5.5-6.5, then heat the hydrolysate to 95°C for 10 minutes to inactivate the enzymes.

[0016] The supernatant membrane separates and retains polypeptides with a molecular weight cutoff of 300-13000 Da, which are then concentrated and dried to obtain bonito active peptide powder.

[0017] Furthermore, the membrane separation method is as follows: the supernatant is ultrafiltered using a 15000 Da ultrafiltration membrane, and the resulting filtrate is then concentrated using a 300 Da nanofiltration membrane.

[0018] The present invention also provides a composition comprising magnesium sulfate and bonito bioactive peptides, wherein the composition has the property of protecting pepsin activity, specifically by improving the thermal stability of pepsin.

[0019] Furthermore, when skipjack tuna active peptides and pepsin (activity 4-12 kDa / g) were mixed at a mass ratio of (2-5):1 and diluted to a mixed enzyme solution with pepsin activity not exceeding 100 u / ml, and then magnesium sulfate was added, the thermal stability and pH stability of the pepsin were significantly improved compared to the pepsin without the addition of the peptides.

[0020] The concentration of magnesium sulfate in the hydrolysate containing pepsin and bonito peptide is 1.0-2.4 g / 100 ml.

[0021] This invention has found that when skipjack tuna active peptides are used as a heat stabilizer in combination with pepsin, the initial pepsin activity should be less than 110 u / ml, and the mass ratio of the peptide to pepsin should be (2-5):1. Within this range, the effect of improving pepsin stability is better. It is possible that the skipjack tuna peptides of the present invention, at a suitable concentration of 300-13000 Da, can be further hydrolyzed by pepsin with suitable activity to generate peptides that protect pepsin (with molecular weights mainly below 5000 Da), thereby improving pepsin stability. When pepsin activity and albumin peptide concentration exceed a certain range, the number of deprotective small peptide products generated by pepsin hydrolysis increases, thus weakening the protective effect of skipjack tuna peptides on pepsin.

[0022] Compared with the prior art, the preparation method and composition of skipjack tuna active peptides provided by the present invention have the following beneficial effects and characteristics:

[0023] The skipjack tuna active peptides provided by this invention not only improve the thermal stability of pepsin and extend its half-life by more than 50%, but also expand the pH activity range of pepsin. After adding skipjack tuna active peptides and magnesium sulfate to pepsin, it can still maintain a certain activity at pH 6-7 and 60-70℃ for 30-40 minutes. Adding a certain amount of magnesium sulfate to the pepsin and skipjack tuna peptide hydrolysate not only has a synergistic effect on improving the stability of pepsin, but also increases the magnesium ion content of the polypeptide hydrolysate, making it a sports nutrition food that supplements peptide proteins and magnesium ions. When not used as a sports nutrition food, excess magnesium ions in the hydrolysate can be removed by nanofiltration. Attached Figure Description

[0024] 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.

[0025] Figure 1 The curves showing the effects of different proportions of skipjack tuna active peptides on porcine pepsin activity provided by this invention;

[0026] Figure 2 The molecular weight distribution diagram of the sample provided for this invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention provides an operational example of a method for preparing skipjack tuna bioactive peptides, the specific steps of which are as follows:

[0029] 1. After removing the internal organs, dry the bonito, grind it into powder, mix it with water at a mass ratio of 1:(20-30), keep it at 60-70℃ for 40-60 minutes to soak and expand, then grind it into a slurry through a colloid mill and filter it through a 100-mesh filter to obtain a slurry;

[0030] 2. Adjust the pH of the slurry to 7.5-8.5, add Aspergillus oryzae protease, and hydrolyze at 38-48℃ for 90-150 minutes to obtain hydrolysate; wherein the amount of Aspergillus oryzae protease added is based on the protein content in the bonito powder, and the enzyme activity is 1000u-2000u per gram.

[0031] 3. After adjusting the pH of the hydrolysate to 5.5-6.5, heat it to 95℃ for 10 minutes to inactivate the enzyme, centrifuge to remove insoluble matter, and obtain the supernatant. Then, ultrafilter the supernatant through a 15000Da ultrafiltration membrane, and concentrate the filtrate through a 300Da nanofiltration membrane to obtain the skipjack tuna active peptide.

[0032] 4. Dry the bonito active peptides into powder to obtain bonito active peptide powder.

[0033] The present invention provides the following embodiments and comparative examples:

[0034] Example 1

[0035] A method for preparing skipjack tuna bioactive peptides is as follows:

[0036] 1. Take 100g of bonito powder (76% protein content), add 2000g of water and mix. Keep it at 60℃ for 60 minutes to allow it to swell. Then grind it into a slurry using a colloid mill and pass the slurry through a 100-mesh filter to obtain the slurry.

[0037] 2. Adjust the pH of the solution to 8.0 with hydrochloric acid, then add 80 kDa of Aspergillus honey protease (Amano Amano 3SD protease), and hydrolyze at 48°C for 90 min to obtain the hydrolysate;

[0038] 3. Adjust the pH of the hydrolysate to 5.5, then heat at 95℃ for 10 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 15000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 300 Da nanofiltration membrane to obtain the concentrated solution of skipjack tuna active peptides.

[0039] 4. The concentrated liquid is freeze-dried to obtain bonito active peptide powder with a molecular weight of 300-13000 Da and a polypeptide content of 85.2%.

[0040] Six portions of porcine pepsin with an activity of 4100 u / g were prepared, each weighing 1 gram. Five portions were mixed with 1 g (A1), 2 g (A2), 3 g (A3), 4 g (A4), and 5 g (A5) of bonito active peptide powder prepared in Example 1, respectively. The remaining portion (A0) was not mixed with bonito active peptide powder. The pure porcine pepsin powder A0 and the other five mixed enzyme powders were dissolved in 100 ml of water, 1.5 g of magnesium sulfate, and 6 g of pea protein isolate to form sample solutions A0, A1, A2, A3, A4, and A5 (the initial activity of porcine pepsin was 41.0 u / ml). The pH was adjusted to 3.0 with hydrochloric acid, and then the samples were heated at 70°C for different times. Samples were taken periodically and cooled to about 0°C in ice water for storage. The porcine pepsin activity of the sample solutions at each time point was then measured (using the national standard Folin-Ciocalteu method), and the half-life T was calculated. 1 / 2 (minutes), results are available. Figure 1 See Table 1.

[0041] Table 1. Activity TL of porcine pepsin solutions with different skipjack tuna peptide addition ratios at 70℃ 1 / 2 With the rate of increase

[0042]

[0043] Note: Half-life T 1 / 2 =0.693 / k;k=(lnc1-lnc2) / t2-t1,t2-t1=-10,c1=41.

[0044] from Figure 1 As shown in Table 1, the original enzyme (A0) without bonito active peptide powder and A1 with bonito active peptide powder added at a 1:1 ratio completely lost their enzyme activity after heating at 70℃ for 15 min. When the ratio of bonito active peptide powder to porcine pepsin was 2:1 (A2), 3:1 (A3), and 4:1 (A4), the samples still showed pepsin activity after 22.5 min, and the pepsin T... 1 / 2 Compared to the original enzyme A0, the activity increased by 100%, 162%, and 230%, respectively; the enzyme activity T was increased by 5:1 (A5). 1 / 2Compared to A0, the ratio increased by 22%, but its protective effect on pepsin was not as good as A2-A4. The reason for this may be that when the added 300-13000 Da bonito peptide comes into contact with porcine pepsin, it hydrolyzes to produce pepsin-protective polypeptides, but may also produce substances that are detrimental to pepsin protection. When the ratio of bonito peptide is appropriate, the protective polypeptides dominate, resulting in a significant protective effect on pepsin. However, when the concentration of bonito peptide is too high, the amount of hydrolysates detrimental to pepsin protection increases, thus weakening the protective effect on pepsin stability. Therefore, the appropriate addition range for bonito peptide to pepsin is (2-5):1.

[0045] Example 2

[0046] A method for preparing skipjack tuna bioactive peptides is as follows:

[0047] 1. Take 100g of bonito powder (protein content 76%), add 2500g of water and mix. Keep it warm at 65℃ for 50 minutes to allow it to swell. Then grind it into a slurry using a colloid mill and pass the slurry through a 100-mesh filter to obtain the slurry.

[0048] 2. Adjust the pH of the solution to 8.5 with hydrochloric acid, then add 100 kDa of Aspergillus honey protease (Amano Amano 3SD protease), and hydrolyze at 40°C for 140 min to obtain the hydrolysate;

[0049] 3. Adjust the pH of the hydrolysate to 6.0, then heat at 95℃ for 10 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 15000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 300 Da nanofiltration membrane to obtain the concentrated solution of skipjack tuna active peptides.

[0050] 4. The concentrated liquid is freeze-dried to obtain bonito active peptide powder with a molecular weight of 300-13000 Da and a polypeptide content of 86.1%.

[0051] Twenty-one portions of bovine pepsin with an activity of 9000 u / g were prepared, each weighing 1 g. Twelve portions were each mixed with 2 g of bonito active peptide powder prepared in Example 2 (denoted as B1), while the other nine portions were not mixed with bonito active peptide powder (denoted as B0). Each of the 21 samples was dissolved in 100 ml of water and 1.4 g of magnesium sulfate (the initial bovine pepsin activity was 90 u / ml for all samples). Six portions of B1 and six portions of B0 were taken, and their pH values ​​were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 respectively using lactic acid and sodium carbonate. The samples were then heated simultaneously in a 60°C water bath for 30 minutes. After heating, the samples were stored at 0°C. The bovine pepsin activity and rate of change under each pH condition were then measured and calculated. The results are shown in Table 2.

[0052] Six additional B1 samples were taken and their pH values ​​were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. Three B0 samples were taken and their pH values ​​were adjusted to 2.0, 3.0, and 4.0, respectively. All nine samples were placed in a 70°C water bath and heated simultaneously for 30 minutes. After the reaction, the samples were quickly placed in ice water and cooled to 0°C for storage. The bovine pepsin activity of the sample solutions was then measured under each pH condition, and the rate of change was calculated. The results are shown in Table 2.

[0053] Table 2. Changes in bovine pepsin activity at different pH levels with different ratios of skipjack tuna peptides.

[0054]

[0055] Table 2 shows that, under the same temperature and pH conditions, the bovine pepsin activity in all B1 samples with added skipjack tuna peptide was significantly higher than that in the B0 sample without skipjack tuna peptide. After heating at 60℃ for 30 min, bovine pepsin remained active in B1 samples at pH 2-7, while it was inactivated in B0 samples at pH above 5. Furthermore, the bovine pepsin activity in B1 samples at pH 2-4 was on average 210.6% higher than that in B0. After heating at 70℃ for 30 min, the bovine pepsin activity in B0 samples was lost at all pH conditions, while the bovine pepsin in B1 samples remained active at pH 2-6. Therefore, skipjack tuna peptide can not only improve the thermal stability of bovine pepsin but also enhance its pH stability and application range.

[0056] Skipjack tuna active peptide powder B prepared in Example 2 was diluted with water to 10 mg / ml and designated as group B0. Another batch of skipjack tuna active peptide powder B was hydrolyzed with pepsin at pH 3 and 60℃ for 30 min. After enzyme inactivation, the supernatant was collected and adjusted to a peptide content of 10 mg / ml, designated as group B1. The molecular weights of the samples in groups B0 and B1 were detected using a UV detector and a G50 gel electrophoresis apparatus. The molecular weight distribution of B0 and B1 was calculated using the normalization method. The results are shown in [Figure number missing]. Figure 2 And Table 3.

[0057] Table 3. Distribution of bonito peptide molecular weight (Da) (%)

[0058]

[0059] from Figure 2As shown in Table 3, the skipjack tuna peptides prepared in Example 2, before hydrolysis with bovine pepsin, contained peptides in liquid B0 with molecular weights less than 13000 Da, with the largest elution peak appearing at 45.8 ml (corresponding to a molecular weight of 7630 Da). When bovine pepsin was added to the skipjack tuna peptide sample, the hydrolysate B1, after hydrolysis at 60°C for 30 minutes, became a mixture of peptides mainly with molecular weights less than 5000 Da, with small peptides with molecular weights less than 3000 Da accounting for more than 68%, and the main elution peak appearing at 91.8 ml (corresponding to a molecular weight of 1950 Da). Therefore, it is believed that after further hydrolysis of the 300-13000 Da skipjack tuna peptides into smaller peptides by bovine pepsin, a certain peptide fragment has a protective effect on the structure of bovine pepsin, thus improving the thermal stability of bovine pepsin.

[0060] Example 3

[0061] A method for preparing skipjack tuna bioactive peptides is as follows:

[0062] 1. Take 100g of bonito powder (protein content 76%), add 3000g of water and mix. Keep it at 70℃ for 40 minutes to allow it to swell. Then grind it into a slurry using a colloid mill and pass the slurry through a 100-mesh filter to obtain the slurry.

[0063] 2. Adjust the pH of the solution to 7.5 with hydrochloric acid, then add 140 kDa of Aspergillus honey protease (Amano Amano 3SD protease), and hydrolyze at 45°C for 120 min to obtain the hydrolysate;

[0064] 3. Adjust the pH of the hydrolysate to 6.0, then heat at 95℃ for 10 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 20000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 300 Da nanofiltration membrane to obtain the concentrated solution of skipjack tuna active peptides.

[0065] 4. The concentrate is freeze-dried to obtain bonito active peptide powder with a molecular weight of 300-13000 Da and a polypeptide content of 84.8%.

[0066] Four samples were taken and designated C1. Each sample was dissolved in 2g of bonito active peptide powder, 1.0g of magnesium sulfate, 6g of bovine collagen powder, and 100ml of purified water, as prepared in Example 3. Four other samples were taken and designated C00. Each sample was dissolved in 100ml of purified water and 6g of bovine collagen powder, without the addition of bonito active peptide powder and magnesium sulfate. After adjusting the pH of each of the eight sample solutions to 3.5, 0.8g of porcine pepsin with an activity of 11ku / g was added to each sample solution (the initial pepsin activity of all eight sample solutions was 88u / ml). C0 and C1 were paired into four pairs and heated in water baths at four different temperatures (40℃, 50℃, 60℃, and 70℃) for 30 minutes each. After heating, the samples were quickly cooled to 0℃ in ice water and stored. The pepsin activity of the samples at each time point was determined using the Folin-Ciocalteu method. The peptide content in the hydrolysate was determined according to GB / T 22492-2008. The calcium and magnesium ion content of the samples hydrolyzed at 40℃ and 50℃ was determined using the BJS201718 method. The results are shown in Table 4.

[0067] Table 4. Decrease rate of enzyme activity and polypeptide content in hydrolysate of skipjack tuna protein by pepsin at different temperatures.

[0068]

[0069] Table 4 shows that after porcine pepsin hydrolyzed bovine collagen at 40-70℃ for 30 min, the polypeptide content in sample C1 with added bonito peptide powder increased by 12.7%-23.3% compared to sample C0 without bonito peptide powder. The higher the temperature, the greater the net increase in polypeptide content, indicating that adding bonito peptide can improve the activity of porcine pepsin by enhancing its thermal stability. After porcine pepsin hydrolyzed bovine collagen at 40-60℃ for 30 min, the pepsin activity decrease rate in sample C1 was 21-23% lower than that in sample C0 (ΔC). Therefore, it can be concluded that when bonito peptide active peptide is mixed with pepsin at a mass ratio of 2.5:1, the pepsin in the solution is protected to a certain extent, and the ability to hydrolyze bovine collagen is significantly improved.

[0070] Analysis of the calcium and magnesium ion content in the hydrolysates of samples C1 and C0 revealed that, due to the protection of pepsin activity in sample C1, collagen hydrolysis was more complete, resulting in higher calcium and magnesium ion content and net increase compared to sample C0 at various temperatures. In this invention, bovine bone collagen enzymatic hydrolysate C1, with the addition of bonito bioactive peptides and magnesium sulfate, exhibits significantly higher levels of easily absorbed peptides, calcium ions, and magnesium ions than conventional pepsin hydrolysates, making it a suitable protein and mineral supplement for athletes.

[0071] Example 4

[0072] A method for preparing skipjack tuna bioactive peptides is as follows:

[0073] 1. Take 100g of bonito powder (protein content 76%), add 3000g of water and mix. Keep it at 70℃ for 40 minutes to allow it to swell. Then grind it into a slurry using a colloid mill and pass the slurry through a 100-mesh filter to obtain the slurry.

[0074] 2. Adjust the pH of the solution to 8.0 with hydrochloric acid, then add 150 kDa of Aspergillus honey protease (Amano Amano 3SD protease), and incubate at 48°C for 90 min to hydrolyze and obtain the hydrolysate;

[0075] 3. Adjust the pH of the hydrolysate to 5.5, then heat at 95℃ for 10 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 20000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 300 Da nanofiltration membrane to obtain the concentrated solution of skipjack tuna active peptides.

[0076] 4. The concentrated liquid is freeze-dried to obtain bonito active peptide powder with a molecular weight of 300-13000 Da and a polypeptide content of 85.2%.

[0077] The protective effect of the skipjack tuna active peptide powder prepared in Example 4 against porcine pepsin was tested.

[0078] Two portions of porcine pepsin with an activity of 4100 u / g, each 0.5 g, were added to 100 ml of water. One portion was dissolved with 1.5 g of bonito active peptide powder (denoted as D1), and the other portion was dissolved without bonito active peptide powder (denoted as D0). 5 g of whey protein powder and 2 g of magnesium sulfate were added to each of the two solutions respectively to prepare sample solutions D0 and D1 (the initial activity of porcine pepsin in both samples was 20.5 u / ml). The pH of the sample solution was adjusted to 4.0 with hydrochloric acid, and then heated at 60℃ for 10 minutes. The sample solutions were then quickly placed in ice water to cool to 0℃ and stored. The porcine pepsin activity C2 of the sample was then determined using the Folin method, and the half-life T was calculated. 1 / 2 (minute).

[0079] Comparative Example 1

[0080] In step 2, the pH of the solution was adjusted to 9.0, and Aspergillus oryzae protease with equal enzyme activity (commercially available) was used to replace the honey-derived protease for hydrolysis. All other steps were the same as in Example 4. The protective effect of the skipjack tuna active peptide powder prepared in Comparative Example 1 against porcine gastric protease was tested using the same method as in Example 4.

[0081] Comparative Example 2

[0082] In step 2, the pH of the solution was adjusted to 7.0, and the honey-derived Aspergillus protease was replaced with papain (commercially available) of equal enzyme activity for hydrolysis. All other steps were the same as in Example 4. The protective effect of the skipjack tuna active peptide powder obtained in Comparative Example 2 against porcine gastric protease was tested using the same method as in Example 4.

[0083] Comparative Example 3

[0084] In step 2, the pH of the solution was adjusted to 3.5, and the Aspergillus oryzae protease with isoenzymatic activity (Shanghai Yuanju Biotechnology Co., Ltd.) was used to replace the Aspergillus honey protease for hydrolysis. The hydrolysis temperature was 55°C, and all other steps were the same as in Example 4. The protective effect of the skipjack tuna active peptide powder prepared in Comparative Example 3 on porcine gastric protease was tested using the same method as in Example 4.

[0085] Comparative Example 4

[0086] In step 2, the pH of the solution was adjusted to 7.5, and Aspergillus oryzae neutral protease with equal enzyme activity (Amano Amano AX protease) was used to replace Aspergillus honey protease for hydrolysis. All other steps were the same as in Example 4. The protective effect of the skipjack tuna active peptide powder prepared in Comparative Example 4 against porcine gastric protease was tested using the same method as in Example 4.

[0087] Comparative Example 5

[0088] In step 2, the pH of the solution was adjusted to 10.0, and the Aspergillus honeysuckle protease was replaced with a commercially available alkaline protease of Bacillus licheniformis with equal enzyme activity for hydrolysis at 60°C. All other steps were the same as in Example 4. The protective effect of the skipjack tuna active peptide powder prepared in Comparative Example 5 against porcine gastric protease was tested using the same method as in Example 4.

[0089] Comparative Example 6

[0090] The preparation method of skipjack tuna active peptides is the same as in Example 4. The difference from Example 4 is that magnesium sulfate is not added to the D0 and D1 sample solutions in the detection method; all other treatments are the same as in Example 4.

[0091] The detection results of Example 4 and Comparative Examples 1-6 are summarized in Table 5 below.

[0092] Table 5. Activity of porcine pepsin in different solutions at 60℃ (T0) 1 / 2

[0093]

[0094] Note: T1 / 2 =0.693 / k; k=(lnc1-lnc2) / t2-t1; t2-t1=10, c1=20.5.

[0095] As can be seen from the statistics in Table 5, the half-life of porcine pepsin activity in all samples of Comparative Examples 1-5 is similar to D0 without the addition of bonito active peptide powder, and is much lower than that in Example 4. This indicates that the hydrolysis products of the proteases used in Comparative Examples 1-5 have essentially no effect on improving the stability of pepsin. The enzyme activity T in Example 4... 1 / 2 T compared to D0 1 / 2The duration was extended by 219.7%. Comparative Example 6, after adding skipjack tuna active peptide powder and pepsin, did not add magnesium sulfate, although the pepsin activity C2 and half-life T... 1 / 2 Both were significantly higher than D0 without the addition of skipjack tuna active peptides, but enzyme activity and T... 1 / 2 Compared to Example 4, the time was shortened by 19.4% and 35.5%. This indicates that using skipjack tuna active peptide powder alone can improve pepsin stability; however, the combined use of skipjack tuna active peptide and magnesium sulfate can further improve pepsin stability, which was an accidental discovery during the experiment, and the mechanism is still unclear. The experiment also found that when the magnesium sulfate concentration is between 1.0-2.4 g / 100 ml, the synergistic effect of skipjack tuna peptide in improving pepsin stability is better; when it deviates from this range, the synergistic effect in improving pepsin stability decreases significantly.

[0096] Our research group compared dozens of experimental cases of hydrolyzing skipjack tuna protein with different combinations of proteases using the same enzymatic hydrolysis and assay methods, as well as experimental cases with different mineral salts added during the enzymatic hydrolysis process. Apart from the combination of peptides obtained by hydrolyzing skipjack tuna with Aspergillus honey and magnesium sulfate, which synergistically improved the stability of pepsin, no other enzyme hydrolysis of skipjack tuna peptides and mineral combinations were found to exert the above synergistic effect.

[0097] In summary, the present invention provides bonito bioactive peptides that have the property of protecting pepsin activity, and can be used as pepsin protectants in the food, agriculture, animal husbandry or feed processing industries, and can also be used as raw materials for sports nutrition foods.

[0098] It should be noted that:

[0099] 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.

[0100] Unless otherwise specified, the raw materials used in this invention, such as porcine pepsin and bovine pepsin, are all existing commercially available products that can be purchased and obtained by those skilled in the art.

[0101] The reagents and instruments used in this embodiment do not specify the manufacturers or other information; they are all conventional products that can be purchased on the market.

[0102] 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 skipjack tuna bioactive peptides, characterized in that: Includes the following steps: Mix bonito powder with water, keep it warm and allow it to swell, then grind it into a slurry using a colloid mill; Honey Aspergillus protease was added to the slurry for hydrolysis to obtain the hydrolysate; After inactivating the enzymes in the hydrolysate and removing impurities by centrifugation, the supernatant is obtained. The supernatant is then separated by membrane separation to retain the polypeptide, thus obtaining the bonito bioactive peptide. The mass ratio of bonito meal to water is 1:(20-30); The temperature for heat preservation and expansion is 60-70℃, and the time is 40-60 minutes; The slurry obtained from the grinding process needs to be sieved through a 100-mesh filter screen; The amount of honey aspergillin added is based on the protein content in the bonito powder, with an enzyme activity of 1000u-2000u per gram. The pH of the slurry is adjusted to 7.5-8.5, the hydrolysis temperature is 38-48℃, and the hydrolysis time is 90-150 min; First, adjust the pH of the hydrolysate to 5.5-6.5, then heat the hydrolysate to 95°C for 10 minutes to inactivate the enzymes. The supernatant was separated by membrane separation to retain active peptides with a molecular weight cutoff of 300-13000 Da, and then concentrated and dried to obtain bonito active peptide powder.

2. The method for preparing skipjack tuna active peptides according to claim 1, characterized in that: The membrane separation method is as follows: the supernatant is ultrafiltered using a 15000Da ultrafiltration membrane, and the filtrate is then concentrated using a 300Da nanofiltration membrane.

3. A composition, characterized in that: Its components include magnesium sulfate and the bonito bioactive peptide of claim 1, and the composition has the property of protecting pepsin activity; The magnesium sulfate concentration is 1.0-2.4 g / 100 ml, and the bonito active peptide is mixed with pepsin at a mass ratio of 2.5:1.

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