Method for preparing milt active peptide by combining compound enzyme treatment with supercritical carbon dioxide extraction

Abstract

The invention belongs to the technical field of polypeptide products, and discloses a method for preparing milt active peptide by combining compound enzyme treatment with supercritical carbon dioxide extraction, which comprises the following steps: respectively combining salmon seminal vesicle homogenate with protease to carry out restrictive enzymolysis reaction; carrying out enzyme deactivation on the obtained enzymatic hydrolysate, centrifuging, taking supernate, and drying the supernate; and performing deodorization treatment on the dry powder by adopting supercritical carbon dioxide extraction to obtain the milt active peptide. Compared with the prior art, the method has the advantages of mild extraction reaction conditions, few side reactions, high extraction efficiency, no damage to amino acid and easiness in control of hydrolysis degree. In addition, flavor optimization is carried out, nutritional ingredients of the protamine active peptide cannot be damaged, the extraction rate is high, the protamine active peptide has excellent rehydration performance and good water solubility, high functional characteristics are reserved, fishy smell and bitter taste are low, oxidation resistance is high, the protamine active peptide is more easily absorbed by the human body, the industrial application field of the protamine is widened, and the protamine active peptide is worthy of popularization and application. A new thought is provided for efficient development of protamine, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to a method for preparing fish sperm active peptides by combining composite enzyme treatment with supercritical carbon dioxide extraction, and belongs to the technical field of active substance extraction. Background Art

[0002] Salmon is a popular fishery resource. However, during the processing, a considerable amount of salmon spermatocysts are usually discarded as industrial waste, causing environmental pollution. Some of them are reused to produce low-value products such as fertilizers and animal feed, and have not been fully developed and utilized. In addition, the spermatocysts of fish such as salmon are microglial and have a strong fishy smell, which is not accepted by consumers, limiting their application in the food industry. However, salmon spermatocysts are rich in nutrients such as protein, nucleic acid and enzymes, and are a potential resource that needs to be developed. Among them, protamine is the most important active ingredient in salmon spermatocysts. It contains a variety of basic amino acids such as arginine and lysine, which can provide sufficient nutrients for the metabolic process. It has health benefits such as anti-diabetes, anti-coagulation, strengthening liver function, reducing oxidative stress and improving male reproductive system function. It also shows a wide range of antioxidant and antibacterial activities and good thermal stability, and can be used as a natural food preservative. At the same time, the rich nucleic acids in salmon spermatocysts also have the effects of improving human immunity and improving sleep quality. At present, the separation efficiency of protamine is low, and there are technical barriers to its efficient application. In addition, due to the strong fishy and bitter taste of this raw material, there is a lack of development and application of protamine, especially the lack of high-value processing in the food industry. Therefore, it is necessary to produce a functional powdered product with high extraction rate and optimized flavor to meet the needs of consumers.

[0003] The extraction of protamine mainly adopts chemical hydrolysis, which has great damage to amino acids, low yield, high cost, and the risk of reagent residue, which limits its application in the food industry. Compared with chemical hydrolysis, enzymatic hydrolysis has the advantages of high polypeptide yield, high efficiency, simple operation, and mild reaction conditions. In addition, it has been reported that the antioxidant properties of polypeptides produced by enzymatic hydrolysis have been improved, and these biologically active polypeptides can be used as active ingredients or materials in food systems. For example, patent document CN117024551A discloses a yellowtail fish essence active peptide and a preparation method thereof. The method includes using alkaline protease to enzymatically hydrolyze yellowtail fish milt homogenate for 1.5h to 2.5h, and then centrifuging, removing the fishy smell with activated carbon, drying and other steps to obtain yellowtail fish essence active peptide. However, in practical applications, different proteases have different action sites. In the above-mentioned technology, only a single enzymatic hydrolysis is used, which usually has the problem of poor effect. Therefore, the present invention intends to develop a method for preparing protamine that can make the obtained enzymatic hydrolysis product have low fishy smell and good water solubility, and have functional properties such as emulsification, foaming, and antioxidant properties, and is suitable for different application scenarios. Summary of the invention

[0004] In order to solve the shortcomings of the existing research and development and application of protamine in food, and overcome the problems of low extraction rate of protamine / strong fishy and bitter taste / poor functional characteristics, the present invention provides a method for preparing protamine active peptides by combining composite enzyme treatment with supercritical carbon dioxide extraction, wherein restricted enzymatic hydrolysis is performed by a combination of proteases, and supercritical carbon dioxide extraction technology is used to remove fishy smell, so as to obtain a protamine active peptide product with optimized flavor (low fishy and bitter taste), high yield and high functionality, which is suitable for industrial application.

[0005] The first technical solution provided by the present invention is a method for preparing protamine active peptides by combining composite enzyme treatment with supercritical carbon dioxide extraction, and the specific steps are as follows:

[0006] (1) Enzymatic hydrolysis: adding a protease combination containing flavor protease to the salmon seminal vesicle homogenate for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0007] (2) Post-enzymatic hydrolysis treatment: inactivate the enzyme in the enzymatic hydrolyzate of step (1), centrifuge and extract the supernatant, and dry it to obtain a dry powder;

[0008] (3) Deodorization: The dry powder of step (3) is subjected to supercritical carbon dioxide extraction to remove the odor and obtain fish essence active peptide.

[0009] In certain embodiments, in step (1), the preparation method of the salmon seminal vesicle homogenate is as follows: fresh salmon seminal vesicles are first washed with purified water, and then soaked and washed with 0.1-0.5M sodium citrate solution for 1-5 times, each time for 0.5 hours. The chopped salmon seminal vesicles are crushed into slurry with a fine grinder, and then the slurry is mixed with purified water in a ratio of 1:1 to 1:5.

[0010] In certain embodiments, in step (1), the protease combination is any one of the complex enzymes (a) to (b):

[0011] (a) neutral protease and flavor protease, the addition ratio of neutral protease to flavor protease is 3:1 to 1:1;

[0012] (b) papain and flavor protease, the added ratio of papain to flavor protease is 3:1 to 1:1;

[0013] (c) trypsin and flavor protease, the addition ratio of trypsin and flavor protease is 3:1 to 1:1;

[0014] (d) Novozym 11028 protease and flavor protease, the added ratio of Novozym 11028 protease and flavor protease is 3:1 to 1:1.

[0015] Furthermore, the protease combinations in (a), (b) and (d) were subjected to enzymolysis at 50°C and pH 7.0, and the protease combination in (c) was subjected to enzymolysis at 37°C and pH 8.0.

[0016] In certain embodiments, in step (1), the enzymatic hydrolysis conditions are as follows: the protease combination is added to the salmon seminal vesicle homogenate, the enzyme / substrate ratio is 3000U / g to 10000U / g, and the enzymatic hydrolysis time is 30 to 120min.

[0017] In certain embodiments, in step (2), the conditions for inactivating the protease after enzymatic hydrolysis are: inactivation temperature 80-95° C., inactivation time 5 min-10 min, and the pH value of each hydrolyzate after inactivation is adjusted to 7.0.

[0018] In certain embodiments, in step (2), the centrifugation and crushing conditions in the post-enzymatic hydrolysis treatment are: centrifugal force 4000g to 8000g, and centrifugation time is not less than 10 min.

[0019] In certain embodiments, in step (2), the centrifugal supernatant is dried, then pulverized in a high-speed pulverizer for 3 to 5 minutes, and passed through a 40 to 60 mesh sieve.

[0020] In certain embodiments, in step (2), the drying conditions in the post-enzymatic hydrolysis treatment are as follows: the enzymatic hydrolysate is dried by spray drying or freeze drying.

[0021] Furthermore, the spray drying operation parameters are as follows: the feed pump controls the feed flow rate to be 80-100 ml / min, the inlet air temperature is 135-170°C, the outlet air temperature is 60-75°C, and the drying end point is reached when the moisture content of the dried product is lower than 3%-5%; the freeze drying operation parameters are as follows: -40°C-80°C, drying for 48-72h, and the drying end point is reached when the moisture content of the dried product is lower than 1%-2%.

[0022] In certain embodiments, in step (3), the conditions for supercritical carbon dioxide extraction are: extraction pressure of 20-35 MPa, extraction temperature of 30-50° C., carbon dioxide flow rate of 10-20 kg / h, and extraction time of 0.5-1.5 h.

[0023] The second technical solution provided by the present invention is a fish essence active peptide prepared by the method described in the first technical solution. The fish essence active peptide is obtained by restrictive enzymolysis using a combination of proteases and deodorization by composite supercritical carbon dioxide extraction. The prepared salmon fish essence active peptide has low fishy smell, high water solubility, and has functional characteristics such as emulsification performance, foaming performance, and antioxidant performance.

[0024] The third technical solution provided by the present invention is the method described in the first technical solution, or the use of the protamine active peptide described in the second technical solution in preparing a protamine active peptide product.

[0025] The technical effects of the present invention are as follows:

[0026] 1. The present invention provides a variety of restriction enzyme hydrolysis conditions, with a high yield of enzymatic hydrolysates and good industrial prospects.

[0027] 2. The water solubility of the protamine active peptide obtained by the present invention is improved by 50% to 98%, and it has good emulsifying properties, foaming properties, antioxidant properties and nutritional characteristics, and can be used as a functional ingredient in the food industry.

[0028] 3. The preparation method provided by the present invention has mild and controllable enzymatic hydrolysis process conditions, simple process, safe ingredients, and releases flavor amino acids through composite enzymatic hydrolysis, thereby reducing the bitterness of the enzymatic hydrolyzate.

[0029] 4. The present invention adopts composite enzymatic hydrolysis in coordination with supercritical carbon dioxide extraction to remove fishy smell, which has obvious fishy smell removal effect without introducing impurities and is safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The protein recovery rate and hydrolysis degree of each protamine active peptide under different enzyme combinations and different enzymatic hydrolysis times;

[0031] Figure 2 The solubility of each protamine active peptide under different enzyme combinations and different enzymatic hydrolysis times;

[0032] Figure 3 The emulsification properties of each protamine active peptide under different enzyme combinations and different enzymatic hydrolysis times;

[0033] Figure 4 The foaming properties of each protamine active peptide under different enzyme combinations and different enzymatic hydrolysis times;

[0034] Figure 5 The antioxidant activity of each protamine active peptide under different enzyme combinations and different enzymatic hydrolysis times;

[0035] Figure 6 This is the state diagram of the sample after the seminal vesicle: water = 2:1 mixture in Comparative Example 3. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0037] Test method:

[0038] 1. Protein recovery rate of protamine active peptide: The protein recovery rate is calculated according to the following formula:

[0039] Protein recovery rate = protein content in enzymatic hydrolysate / protein content in raw material * 100%.

[0040] 2. The hydrolysis degree of fish sperm active peptide was determined by o-phthalaldehyde (OPA) method.

[0041] 3. Solubility of fish sperm active peptide: Prepare fish sperm active peptide into a 1% solution, centrifuge at 8000g to obtain the supernatant. Determine the protein content of the supernatant and the whole solution respectively. The protein content is determined by the biuret method. The solubility is calculated according to the following formula: Solubility = protein content in supernatant / protein content in stock solution * 100%.

[0042] 4. Emulsification: The emulsification activity and emulsification stability were determined by turbidity method. 10 mg / mL fish essence active peptide solution and rapeseed oil were mixed in a ratio of 3:1, and emulsified for 2 minutes at 13500 r / min using Ultra-Turrax18 high-speed shear disperser. The prepared emulsion was quickly poured into a 25 mL small beaker, and 20 μL of the emulsion was immediately taken from 5 mm from the bottom of the beaker into a test tube. 5 mL of 0.1% SDS solution was added to the test tube, and the absorbance value (A0) was measured at 500 nm after mixing; the sample was re-sampled and the absorbance value (A) was measured after standing for 30 minutes. t ) 0.1% SDS solution was used as blank control.

[0043] Emulsifying activity: EAI(m 2 / g)=2×2.303×A0×N / [C×(1-Φ)×10 4 ];

[0044] Emulsion stability: ESI (m 2 / g)=(A t / A0)×100,

[0045] Where: N: dilution factor, C: protein concentration in the protein aqueous solution before emulsion formation (g / mL)

[0046] Φ: volume fraction of oil in the emulsion (L / L).

[0047] 5. Foaming property: Pour 100 mL of 10 mg / mL protein solution into a 500 mL measuring cylinder and shear for 1 min at 17500 rpm / min using an Ultra-Turrax 18 high-speed shear disperser. Record the foam height as V0. After standing for 30 min, record the foam height again as V 30 .

[0048] Foaming ability (%) = (V0-100) / 100*100%;

[0049] Foam stability (%) = (V 30 -100) / (V0-100)*100%.

[0050] 6. Antioxidant activity: Antioxidant activity is characterized by the ABTS free radical scavenging ability. 7mmol / L ABTS and 2.4mmol / L potassium persulfate are reacted at room temperature in the dark for 12-16h to obtain ABTS stock solution. When measuring the sample, it is diluted with sodium phosphate buffer (10mmol / L, pH 7.4) to obtain ABTS working solution (absorbance at 734nm is 0.70±0.02). 20μL test sample and 1980μL ABTS working solution are reacted at room temperature in the dark for 10min, and then the absorbance at 734nm is measured and recorded as As, and the diluted working solution is recorded as A0. Free radical scavenging ability (%) = (A0-A s ) / A0*100%.

[0051] 7. Sensory evaluation: 20 sensory assessors were selected, aged between 20 and 30, with no sensory defects and good odor recognition and description abilities. They were classified and evaluated according to the sensory evaluation comprehensive scoring method, with grades 1 to 9 (1 for the worst and 9 for the best. Taking fishy smell as an example, 1 means a strong fishy smell that is unacceptable, and 9 means minimal or no fishy smell that does not affect the flavor of the product). The final odor value was the average of 20 replicates.

[0052] The raw materials used in the examples are:

[0053] The neutral protease, Novozym 11028 protease and flavor protease used were purchased from Novozymes, and papain was purchased from Nanning Pangbo Bioengineering Co., Ltd. The enzyme activity of the protease used in the present invention is defined as follows: Under the optimal pH and temperature conditions of the protease, 1 mL of the diluted sample enzyme solution hydrolyzes casein to produce 1 μg of tyrosine per minute, which is one enzyme activity unit (U / g). The enzyme activity of the protease used in the present invention is determined as follows: Trypsin (2.0×10 5 U / g), papain (3.0×10 5 U / g), neutral protease (1.0×10 5 U / g), Novozym 11028 protease (2.0×10 5 U / g).

[0054] Example 1

[0055] The specific steps are as follows:

[0056] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.3M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0057] (2) Add neutral protease and flavor protease to the salmon seminal vesicle homogenate obtained in step (1). The ratio of neutral protease to flavor protease is 3:1, 2:1 and 1:1 respectively. The enzyme / substrate ratio is 6000 U / g. The sample is enzymatically hydrolyzed at 50°C and pH 7.0 for 120 min.

[0058] (3) After enzymatic hydrolysis, the samples were treated at 95°C for 5 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0059] The obtained supernatant is spray dried with the following operating parameters: the feed pump controls the feed flow rate to 80-100 ml / min, the inlet air temperature to 135-170°C, the outlet air temperature to 60-75°C, and the drying endpoint is reached when the moisture content of the dried product is lower than 3%-5%.

[0060] The dried product was crushed by a high-speed crusher for 3 minutes and finally passed through a 40-mesh sieve.

[0061] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 25 MPa, extraction temperature of 50°C, carbon dioxide flow rate of 20 kg / h, and extraction time of 1.5 h.

[0062] The protein recovery rate, hydrolysis degree and sensory evaluation were performed on the active peptide obtained in Example 1. The results are shown in Table 1. Compared with the active peptide obtained under the condition that the addition amount of neutral protease and flavor protease was 2:1, the bitterness optimization effect of the protamine active peptide obtained under the condition of addition amount ratio of 3:1 was limited (the sensory evaluation bitterness acceptability was 4.7), and under the condition of addition amount ratio of 1:1, the protein recovery rate and hydrolysis degree were low (28% and 22%, respectively), and finally it was determined that the optimal ratio of the addition amount of the two enzymes in the protease combination was 2:1.

[0063] Table 1 Sensory evaluation results of fish sperm active peptides under different enzyme ratios

[0064] Dual enzyme complex ratio Protein recovery rate (%) Degree of hydrolysis (%) bitterness Fishy smell 1：1 28％ 22％ 7.83±0.56 7.67±0.84 2：1 47％ 34％ 7.15±0.98 7.35±0.91 3：1 51％ 40％ 4.7±0.55 6.21±0.81

[0065] Example 2

[0066] The specific steps are as follows:

[0067] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.5 M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0068] (2) Neutral protease and flavor protease were added to the salmon seminal vesicle homogenate obtained in step (1), and the ratio of neutral protease to flavor protease was 2:1. The enzyme / substrate ratios were 3000U / g, 6000U / g, 9000U / g and 10000U / g, respectively. The sample was enzymatically hydrolyzed at 50°C and pH 7.0 for 120min.

[0069] (3) After enzymatic hydrolysis, the samples were treated at 80°C for 10 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0070] The obtained supernatant was freeze-dried with the following operating parameters: -80°C, drying for 48 hours, and the drying endpoint was reached when the moisture content of the dried product was less than 2%.

[0071] The dried product was crushed by a high-speed crusher for 5 minutes and finally passed through a 60-mesh sieve.

[0072] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 30 MPa, extraction temperature of 40°C, carbon dioxide flow rate of 15 kg / h, and extraction time of 1.0 h.

[0073] The protein recovery rate and hydrolysis degree of the active peptide obtained in Example 2 were measured. The results are shown in Table 2. Compared with the enzyme / substrate ratio of 3000U / g, the protein recovery rate and hydrolysis degree of the active peptide obtained under the enzyme / substrate ratio of 6000U / g were significantly increased (P<0.05). Compared with the active peptide obtained under the enzyme / substrate ratio of 6000U / g, the protein recovery rate and hydrolysis degree of the protamine active peptide obtained under the condition of 9000U / g were not significantly improved (P>0.05). From the perspective of cost saving in industry, the optimal enzyme / substrate ratio was finally determined to be 6000U / g.

[0074] Table 2 Protein recovery and hydrolysis degree at different enzyme / substrate ratios

[0075] Enzyme / substrate ratio Protein recovery rate (%) Degree of hydrolysis (%) 3000U / g <![CDATA[22.0±2.0 c ]]> <![CDATA[22.7±2.1 a ]]> 6000U / g <![CDATA[47.0±1.0 b ]]> <![CDATA[34.7±1.5 b ]]> 9000U / g <![CDATA[49.0±1.0 ab ]]> <![CDATA[36.0±1.0 b ]]> 10000U / g <![CDATA[50.3±1.5 a ]]> <![CDATA[37.0±1.1 b ]]>

[0076] Note: a, b, c represent the significance of the differences among the groups.

[0077] Example 3

[0078] The specific steps are as follows:

[0079] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.5 M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0080] (2) Neutral protease and flavor protease were added to the salmon seminal vesicle homogenate obtained in step (1), and the ratio of neutral protease to flavor protease was 2:1. The enzyme / substrate ratio was 6000 U / g. The sample was enzymatically hydrolyzed at 50°C and pH 7.0 for 120 min. The sample was sampled and tested every 30 min from the start of the enzymatic hydrolysis.

[0081] (3) After enzymatic hydrolysis, the samples were treated at 80°C for 10 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0082] The obtained supernatant was freeze-dried with the following operating parameters: -80°C, drying for 48 hours, and the drying endpoint was reached when the moisture content of the dried product was less than 2%.

[0083] The dried product was crushed by a high-speed crusher for 5 minutes and finally passed through a 60-mesh sieve.

[0084] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 30 MPa, extraction temperature of 40°C, carbon dioxide flow rate of 15 kg / h, and extraction time of 1.0 h.

[0085] Example 4

[0086] The specific steps are as follows:

[0087] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.5M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0088] (2) Papain and flavor protease were added to the salmon seminal vesicle homogenate obtained in step (1), and the ratio of papain to flavor protease was 2:1. The enzyme / substrate ratio was 6000 U / g. The sample was enzymatically hydrolyzed at 50°C and pH 7.0 for 120 min. The sample was sampled and tested every 30 min from the start of the enzymatic hydrolysis.

[0089] (3) After enzymatic hydrolysis, the samples were treated at 80°C for 10 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0090] The obtained supernatant was freeze-dried with the following operating parameters: -80°C, drying for 48 hours, and the drying endpoint was reached when the moisture content of the dried product was less than 2%.

[0091] The dried product was crushed by a high-speed crusher for 5 minutes and finally passed through a 60-mesh sieve.

[0092] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 30 MPa, extraction temperature of 40°C, carbon dioxide flow rate of 15 kg / h, and extraction time of 1.0 h.

[0093] Example 5

[0094] The specific steps are as follows:

[0095] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.5M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0096] (2) Trypsin and flavor protease were added to the salmon seminal vesicle homogenate obtained in step (1), and the ratio of trypsin to flavor protease was 2:1. The enzyme / substrate ratio was 6000 U / g. The sample was enzymatically hydrolyzed at 37° C. and pH 8.0 for 120 min.

[0097] (3) After enzymatic hydrolysis, the samples were treated at 80°C for 10 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0098] The obtained supernatant was freeze-dried with the following operating parameters: -80°C, drying for 48 hours, and the drying endpoint was reached when the moisture content of the dried product was less than 2%.

[0099] The dried product was crushed by a high-speed crusher for 5 minutes and finally passed through a 60-mesh sieve.

[0100] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 30 MPa, extraction temperature of 40°C, carbon dioxide flow rate of 15 kg / h, and extraction time of 1.0 h.

[0101] Example 6

[0102] The specific steps are as follows:

[0103] (1) Fresh salmon seminal vesicles were first washed with purified water, then soaked and washed with 0.5M sodium citrate solution for 3 times, each time for 0.5 h. The chopped salmon seminal vesicles were crushed into slurry with a fine grinder, and then the slurry was mixed with purified water in a ratio of 1:2.

[0104] (2) Novozym 11028 protease and flavor protease were added to the salmon seminal vesicle homogenate obtained in step (1), and the addition ratio of Novozym 11028 protease to flavor protease was 2:1. The enzyme / substrate ratio was 6000 U / g. The sample was enzymatically hydrolyzed at 50° C. and pH 7.0 for 120 min.

[0105] (3) After enzymatic hydrolysis, the samples were treated at 80°C for 10 min to inactivate the protease, and the pH value of each hydrolyzate was adjusted to 7.0. The centrifugal force was 8000 g and the centrifugation time was 10 min.

[0106] The obtained supernatant was freeze-dried with the following operating parameters: -80°C, drying for 48 hours, and the drying endpoint was reached when the moisture content of the dried product was less than 2%.

[0107] The dried product was crushed by a high-speed crusher for 5 minutes and finally passed through a 60-mesh sieve.

[0108] (4) The dry powder obtained above was subjected to supercritical carbon dioxide extraction to remove the fishy smell. The specific conditions were: extraction pressure of 30 MPa, extraction temperature of 40°C, carbon dioxide flow rate of 15 kg / h, and extraction time of 1.0 h.

[0109] The protein recovery rate, hydrolysis degree, water solubility, emulsification and foaming properties, and antioxidant properties of the protamine active peptides obtained in Examples 3 to 6 were tested, and the results confirmed the feasibility of the protamine active peptides as functional food ingredients.

[0110] like Figure 1 As shown, the active peptide obtained in Example 5, namely the combined enzymatic hydrolysis group of trypsin and flavor protease, showed the highest protein recovery rate and hydrolysis degree, reaching 82.1% and 41.7% respectively.

[0111] like Figure 2 As shown, the solubility before enzymolysis is about 20%. Compared with the sample before enzymolysis, the water solubility of the fish sperm active peptide is greatly improved (50% to 98%). Good water solubility provides the application basis of the fish sperm active peptide.

[0112] like Figure 3 As shown in Examples 4 to 6, the active peptide obtained in Example 4, namely the composite enzymatic hydrolyzate of papain and flavor protease, has the highest emulsification stability (87.7%) and is suitable for food emulsification systems. Figure 4As shown, the active peptide obtained in Example 5 has the highest foam stability (66.5%) and is suitable for food foam systems.

[0113] like Figure 5 As shown, the active peptide obtained in Example 6 exhibited similar antioxidant potential (free radical scavenging ability) as the active peptide obtained in Example 3. The results showed that the free radical scavenging ability of the active peptide obtained in Example 6 reached 94.8%. The results of Examples 3 to 6 confirmed the feasibility of protamine active peptide as a functional food ingredient.

[0114] comprehensive Figures 3 to 5 Compared with the active peptides obtained in Examples 4 to 6, the active peptide obtained in Example 3 showed the highest emulsifying activity and foaming ability, reaching 7.1 m 2 / g, and the foaming capacity reached 43.4%. In addition, the active peptide obtained in Example 3 showed a strong antioxidant potential (free radical scavenging ability), and its free radical scavenging ability reached 95.9%.

[0115] Comparative Example 1

[0116] Referring to steps (1) to (3) of Example 1, the supercritical carbon dioxide extraction step of step (4) in Example 1 is omitted, wherein in step (2), the ratio of the addition amount of neutral protease to flavor protease is 2:1, and other conditions or parameters are consistent with those in Example 1.

[0117] Comparative Example 2

[0118] The composite enzymatic hydrolysis in Example 1 was replaced by single enzymatic hydrolysis, and (a) neutral protease, (b) papain, (c) trypsin, and (d) Novozym 11028 protease were added in step (2), respectively. Other conditions or parameters were the same as those in Example 1.

[0119] In order to verify the effect of this method on the flavor optimization of fish protein active peptide, the samples obtained in comparative examples 1 and 2 and the samples obtained in examples 3 to 6 were subjected to sensory evaluation. After testing, the sensory evaluation results of the smell, bitterness and overall acceptability of the fish protein active peptides (1%) obtained in examples 3 to 6 of the present invention and comparative examples 1 to 2 are shown in Table 3. Compared with comparative examples 1 to 2, the overall acceptability of examples 3 to 6 was significantly improved, and the fishy and bitter taste was reduced. Among them, the fish protein active peptide obtained in example 6 had the lowest fishy and bitter taste and the best overall flavor. Comparative example 1 had the highest fishy smell, and comparative example 2 had the highest bitter taste, which respectively confirmed the effects of composite enzymatic hydrolysis and supercritical carbon dioxide deodorization. The results show that composite enzymatic hydrolysis in conjunction with supercritical carbon dioxide extraction deodorization is a feasible and effective method for flavor optimization.

[0120] Table 3 Sensory evaluation results of each protamine active peptide in Examples 3 to 6, Comparative Examples 1 and 2

[0121] Grouping Fishy smell bitterness Overall acceptability Comparative Example 1 1.90±0.81 4.10±0.91 2.95±0.94 Comparative Example 2(a) 4.05±0.66 2.95±0.57 3.25±0.83 Comparative Example 2(b) 4.35±0.73 3.05±0.35 4.95±0.21 Comparative Example 2(c) 4.40±0.57 3.00±0.44 3.90±0.88 Comparative Example 2(d) 5.10±0.89 3.40±0.59 4.85±0.91 Example 3 7.35±0.91 7.15±0.98 7.00±0.74 Example 4 6.40±0.87 6.60±0.98 6.65±0.74 Example 5 6.45±0.94 6.55±0.81 6.35±0.81 Example 6 7.70±0.66 7.65±0.67 7.45±0.60

[0122] Comparative Example 3

[0123] (1) The chopped salmon spermatocysts are crushed into a slurry using a fine grinder, and then the slurry is mixed with purified water in a ratio greater than 1:1. Due to the special nature of salmon spermatocysts as a raw material, the ratio is greater than 1:1 (e.g. Figure 6 As shown in the figure, there will be a problem of excessive viscosity, which will hinder the next enzymatic hydrolysis operation.

[0124] (2) Since salmon seminal vesicle homogenate has a high viscosity, it is impossible to perform supercritical carbon dioxide deodorization treatment before enzymatic hydrolysis. Therefore, in the method described in the present invention, composite enzymatic hydrolysis must be performed first, and then supercritical carbon dioxide extraction must be performed to remove the fishy smell.

[0125] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing protamine active peptide, characterized in that: The steps include: (1) Enzymatic hydrolysis: adding a protease combination containing flavor protease to the salmon seminal vesicle homogenate for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; (2) Post-enzymatic hydrolysis treatment: inactivate the enzyme in the enzymatic hydrolyzate of step (1), centrifuge and extract the supernatant, and dry it to obtain a dry powder; (3) Deodorization: The dry powder of step (3) is subjected to supercritical carbon dioxide extraction to remove the odor and obtain fish essence active peptide.

2. The method according to claim 1, characterized in that In step (1), the preparation method of the salmon seminal vesicle homogenate is as follows: the salmon seminal vesicle is washed and then soaked and washed with 0.1-0.5M sodium citrate solution for 1-5 times, each time for 0.5 hour, then the salmon seminal vesicle is chopped and crushed into slurry, and then the slurry is mixed with pure water in a ratio of 1:1 to 1:

5.

3. The method according to claim 1 or 2, characterized in that: In step (1), the protease combination is any one of the complex enzymes (a) to (b): (a) neutral protease and flavor protease, the addition ratio of neutral protease to flavor protease is 3:1 to 1:1; (b) papain and flavor protease, the added ratio of papain to flavor protease is 3:1 to 1:1; (c) trypsin and flavor protease, the addition ratio of trypsin and flavor protease is 3:1 to 1:1; (d) Novozym 11028 protease and flavor protease, the added ratio of Novozym 11028 protease and flavor protease is 3:1 to 1:1; Optionally, the protease combination in (a), (b) and (d) is subjected to enzymolysis at 50°C, pH 7.0, and the protease combination in (c) is subjected to enzymolysis at 37°C, pH 8.

0.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), the enzymatic hydrolysis conditions are as follows: the protease combination is added to the salmon seminal vesicle homogenate, the enzyme / substrate ratio is 3000U / g to 10000U / g, and the enzymatic hydrolysis time is 30 to 120min.

5. The method according to any one of claims 1 to 4, characterized in that: In step (2), the conditions for inactivating the protease after enzymatic hydrolysis are: inactivation temperature 80-95° C., inactivation time 5 min-10 min, and the pH value after inactivation is adjusted to 7.

0.

6. The method according to any one of claims 1 to 5, characterized in that: In step (2), the centrifugal and pulverizing conditions in the enzymatic post-treatment are: centrifugal force 4000g to 8000g, and the centrifugal time is not less than 10min; After the centrifugal supernatant is dried, it is crushed by a high-speed crusher for 3 to 5 minutes and passed through a 40 to 60 mesh sieve.

7. The method according to any one of claims 1 to 6, characterized in that: In step (2), the drying conditions in the post-enzymatic hydrolysis treatment are as follows: the enzymatic hydrolysate is dried by spray drying or freeze drying; Optionally, the spray drying operation parameters are: the feed pump controls the feed flow rate to be 80-100 ml / min, the inlet air temperature is 135-170°C, the outlet air temperature is 60-75°C, and the drying end point is reached when the moisture content of the dried product is lower than 3%-5%; the freeze drying operation parameters are: -40°C-80°C, drying for 48-72h, and the drying end point is reached when the moisture content of the dried product is lower than 1%-2%.

8. The method according to any one of claims 1 to 7, characterized in that: In step (3), the conditions of supercritical carbon dioxide extraction are: extraction pressure of 20-35 MPa, extraction temperature of 30-50° C., carbon dioxide flow rate of 10-20 kg / h, and extraction time of 0.5-1.5 h.

9. Protamine active peptide prepared by the method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 8, or use of the protamine active peptide according to claim 9 in preparing a protamine active peptide product.

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