A preparation method of high-purity fish skin collagen tripeptide

Abstract

The invention discloses a method for preparing a high-purity fish skin collagen tripeptide, and belongs to the field of food technology. The present invention is to solve the problems of large usage of composite enzymes, residual salt and free amino acids in the product, and low yield and purity of collagen tripeptides in the existing collagen tripeptide production process, and proposes to use composite enzyme ladder enzymolysis and ultrafiltration membrane to jointly prepare small molecule fish skin collagen peptides. Specifically, by neutral protease and papain ladder enzymolysis, the collagenase hydrolysis efficiency is maximized, and then papain that can specifically hydrolyze glycine (Gly) end is used to deeply hydrolyze 300-1000Da oligopeptides to improve the yield of collagen tripeptides; ultrafiltration and nanofiltration membrane combined separation technology are used to further purify collagen peptides, and free amino acids and peptides with molecular weight greater than 700Da are removed to improve collagen tripeptide (about 300Da) purity.

Description

Technical Field

[0001] The invention belongs to the technical field of food, and particularly relates to a method for preparing high-purity fish skin collagen tripeptide. Background Art

[0002] In recent years, the proportion of collagen peptides in the international market has increased rapidly. The overall annual sales volume of collagen peptides worldwide has reached 25,000 tons, and it has formed multiple application fields such as beauty, joint and bone health, sports health, and external products. Collagen tripeptide is a small peptide containing three amino acids obtained by hydrolysis of collagen or gelatin, which has the characteristics of easy absorption. There is a peptide transporter PepT 1 in the human intestine, which can specifically transport dipeptides and tripeptides. Therefore, collagen tripeptide can be directly absorbed by the intestine or absorbed by the body after incomplete digestion. In addition, a large number of studies have shown that collagen tripeptide has strong physiological function activity, such as promoting skin and bone repair, promoting mineral element absorption, and anti-oxidation. Collagen tripeptide can be used as a nutritional supplement or food fortifier, etc., to regulate human functions through oral administration; it can also be used in cosmetics to improve skin moisture and elasticity through transdermal absorption.

[0003] For collagen peptides, the yield and purity of small molecule peptides, especially tripeptides, are key indicators that determine their functional activity. The invention patent application with application number 202111305526.7 proposes a method for producing collagen tripeptides for skin repair, in which a multi-stage radiation filter cartridge is used to remove impurities from fish skin, and then a composite protease, flavor protease and alkaline protease are used in a 1:1:1 composite enzymatic hydrolysis to obtain collagen tripeptides. However, this method uses a one-time addition of a composite enzyme for enzymatic hydrolysis, and the amount of enzyme used is large. In addition, the non-specific enzymatic hydrolysis products are not further separated to remove free amino acids and undegraded macromolecular peptides, so there is a problem of low purity of the tripeptide. The invention patent application with publication number CN113698472A proposes a method for preparing a high-purity small molecule fish skin collagen peptide and a fish skin collagen peptide spray. The method mainly uses pepsin and alkaline protease to hydrolyze collagen, and then uses dextran gel to purify small molecule peptides, but the small molecule peptides defined in the method refer to hexapeptides with a molecular weight of <700Da and smaller peptides, and the selected complex enzyme needs to use a large amount of acid-base reagents to adjust the pH value, resulting in a large amount of salt residue in the product, and the dextran gel chromatography column is difficult to use for industrial production. It can be seen from this that a method for producing collagen tripeptides with low complex enzyme usage, high product yield and purity is urgently needed. Summary of the invention

[0004] In order to solve the problems of large amount of compound enzyme used, residual salt and free amino acids in the product, low yield and purity of collagen tripeptide in the existing collagen tripeptide production process, the present invention adopts the combined technology of step-by-step compound enzymatic hydrolysis and membrane separation to prepare high-purity fish skin collagen tripeptide. This method can effectively improve the yield and purity of collagen tripeptide, and minimize the amount of compound enzyme used, and reduce the residual salt and free amino acids in the product. The specific technical scheme of the present invention is as follows:

[0005] The present invention provides a method for preparing high-purity fish skin collagen tripeptide, comprising the following steps:

[0006] (1) Fish skin gelatin swelling;

[0007] (2) enzymolyzing the swollen fish skin gelatin using a neutral protease to obtain a fish skin gelatin hydrolyzate;

[0008] (3) enzymatically hydrolyzing the fish skin gelatin hydrolyzate obtained in step (2) using papain to obtain a fish skin gelatin hydrolyzate;

[0009] (4) filtering the fish skin gelatin hydrolyzate obtained in step (3) using an ultrafiltration membrane, and collecting the effluent to remove the high molecular weight peptides with a molecular weight of >1 kDa that are not fully hydrolyzed;

[0010] (5) filtering the effluent obtained in step (4) by the first step of nanofiltration, collecting the effluent to separate the fish skin collagen tripeptide;

[0011] (6) filtering the effluent obtained in step (5) by a second nanofiltration step to remove free amino acids and salts mixed in the fish skin collagen tripeptide;

[0012] (7) Low temperature pasteurization;

[0013] (8) Freeze drying.

[0014] In one embodiment of the present invention, the swelling in step (1) is to prepare the fish skin gelatin into a solution with a mass fraction of 5%-10%, and heat it at 80-100° C. for 10 minutes.

[0015] In one embodiment of the present invention, the amount of the neutral protease added in step (2) is 5000-8000 U / g, the enzymolysis temperature is 50-65° C., and the enzymolysis time is 2-3 h.

[0016] In one embodiment of the present invention, it is characterized in that the amount of papain added in step (3) is 2000-3000 U / g, the enzymolysis temperature is 50-65° C., and the enzymolysis time is 1.5-2 h.

[0017] In one embodiment of the present invention, the ultrafiltration membrane in step (4) has a molecular weight cutoff of 1-2 kDa and a membrane concentration factor of 4-5 times.

[0018] In one embodiment of the present invention, the molecular weight cutoff of the nanofiltration membrane in the first nanofiltration in step (5) is 800-1000Da, and the concentration factor is 4-5 times.

[0019] In one embodiment of the present invention, the nanofiltration membrane molecular weight cutoff of the second nanofiltration in step (6) is 260Da, the concentration multiple is 3-4 times, and after concentration, 1-2 times the volume of water is added to wash out the free amino acids and salts.

[0020] The present invention also provides a fish skin collagen tripeptide obtained by the preparation method.

[0021] Beneficial effects of the present invention:

[0022] In order to solve the problems of large amount of composite enzyme used, residual salt and free amino acids in the product, low yield and purity of collagen tripeptide in the existing collagen tripeptide production process, the present invention proposes to use composite enzyme cascade enzymatic hydrolysis and ultrafiltration membrane to prepare small molecule fish skin collagen peptide. The specific process advantages are reflected in the following aspects: ① Through the cascade enzymatic hydrolysis of neutral protease and papain, the most of the peptide segments are first hydrolyzed with a strong neutral protease, so that the collagenase site in the collagen peptide is fully exposed, and the collagenase hydrolysis efficiency is maximized, and then the papain that can specifically hydrolyze the glycine (Gly) end is used to deeply hydrolyze the 300-1000Da oligopeptide to improve the yield of collagen tripeptide; ② The collagen peptide is further purified by ultrafiltration and nanofiltration membrane combined separation technology to remove free amino acids and peptides with a molecular weight greater than 700Da to improve the purity of collagen tripeptide (about 300Da). DETAILED DESCRIPTION

[0023] The technical solutions described in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The neutral protease and papain used in the present invention are both commercially available proteases.

[0025] The method for detecting the hydrolysis rate of fish skin collagen tripeptide involved in the present invention is as follows:

[0026] 1. Preparation of OPA reagent (prepare and use immediately, store for up to 7 days)

[0027] ① 0.2g OPA (o-phthalaldehyde) + 5mL anhydrous ethanol, mix and dissolve, and place with tin foil to avoid light;

[0028] ②4.767g sodium tetraborate, 0.25g SDS, 0.22g DTT (dithiothreitol), add 150mL deionized water to dissolve. Mix ①② and put into a 250mL brown volumetric flask.

[0029] 2. Drawing of standard curve

[0030] 0.0328 g of L-leucine was added to a 250 mL volumetric flask to prepare a 1 mmol / L leucine solution;

[0031] Take 0, 2, 4, 6, 8, and 10 mL of the prepared 1 mmol / L leucine standard sample, dilute to 10 mL with deionized water to make the concentrations 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L, respectively. Then, take 0.3 mL and add 3 mL of OPA reagent. Shake well and react for 2 minutes in the dark. Immediately measure the OD value at 340 nm using a UV spectrophotometer.

[0032] 3. Measurement

[0033] The sample was pre-diluted to ensure a concentration between 0.5-1 mg / mL;

[0034] Add 3 mL of the prepared OPA reagent to a 4 mL brown centrifuge tube, then add 0.3 mL of the sample to be tested, shake well, react for 2 minutes in the dark, and immediately measure the OD value at 340 nm using a UV spectrophotometer.

[0035] 4. Calculation of hydrolysis rate results

[0036]

[0037] β 明胶 =0.457,α 明胶 =0.796, h tot明胶 =11.1

[0038]

[0039] Wherein, X is the mass of the sample in g; P is the protein content of the sample.

[0040] The calculation method of the fish skin collagen tripeptide yield of the present invention is as follows:

[0041] Yield = fish skin collagen tripeptide mass ÷ total fish skin collagen mass before hydrolysis × 100%

[0042] The tripeptide purity detection method of the present invention is as follows:

[0043] The molecular weight distribution of the peptides in the obtained tripeptide samples was determined using high performance liquid chromatography (HPLC).

[0044] Sample processing: Adjust the pH of the sample to about 7, then filter it through a 0.45 μm filter membrane and place it in a sample injection vial.

[0045] Chromatographic conditions: The gel chromatography column is TSKgel, the mobile phase is 30% acetonitrile + 70% ultrapure water + 0.07% trifluoroacetic acid solution, degassed with a vacuum pump, the ultraviolet detection wavelength is 214nm; the column temperature is 25°C, the flow rate is 1mL / min, and the injection volume is 10μL.

[0046] The elution time of the tripeptide was determined using different molecular weight standards, and the purity of the tripeptide was expressed as the ratio of the peak area at the elution time to the total peak area.

[0047] Embodiment 1:

[0048] (1) Fish skin gelatin swelling: The fish skin gelatin was prepared into a solution with a mass fraction of 8%, and heated at 90° C. for 10 minutes to promote the full swelling of the gelatin;

[0049] (2) First step of enzymatic hydrolysis: using neutral protease to perform one-step enzymatic hydrolysis on the dissolved fish skin gelatin, the enzyme addition amount is 5000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis time is 2.5h;

[0050] (3) Second step of enzymatic hydrolysis: After the enzymatic hydrolysis by neutral protease, the fish skin gelatin hydrolysate was subjected to secondary enzymatic hydrolysis using an appropriate amount of papain, the enzyme addition amount was 2500 U / g, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 1.5 h;

[0051] (4) Ultrafiltration membrane filtration: The fish skin gelatin hydrolyzate after two-step enzymatic hydrolysis is subjected to ultrafiltration membrane filtration to remove the high molecular weight peptides with a molecular weight>1 kDa that are not fully hydrolyzed. The membrane cutoff molecular weight used is 2 kDa, the membrane concentration multiple is 4 times, and the effluent is collected;

[0052] (5) First step: Nanofiltration membrane filtration: The collected ultrafiltration effluent mainly contains oligopeptides with a molecular weight of <2 kDa. The oligopeptide mixture is further separated using a nanofiltration membrane. A nanofiltration membrane with a molecular weight cutoff of 1000 Da is selected to separate the tripeptides, with a concentration factor of 4. The tripeptides are mostly distributed in the effluent;

[0053] (6) Second step: Nanofiltration: The effluent collected in the first step of nanofiltration is mainly tripeptides, free amino acids and salts. The purpose of this step is to remove the free amino acids and salts mixed in the tripeptides. A nanofiltration membrane with a molecular weight cutoff of 260 Da is selected for treatment, the concentration multiple is 4 times, and then 2 times of water is added to further wash out the free amino acids and salts;

[0054] (7) Low-temperature pasteurization: low-temperature pasteurization of the concentrated solution obtained in step (6);

[0055] (8) Freeze-drying: freeze-dry the sterilized fish skin collagen tripeptide concentrate obtained in step (7) to obtain a high-purity fish skin collagen tripeptide powder.

[0056] In this embodiment, the final yield of high-purity tripeptide was measured to be 68.6%, of which the purity of the tripeptide was 85.9%, the hydrolysis rate was 31.9%, the product powder was fine, the saltiness was moderate, and there was no obvious bitter smell.

[0057] Embodiment 2:

[0058] (1) Fish skin gelatin swelling: The fish skin gelatin was prepared into a solution with a mass fraction of 8%, and heated at 90° C. for 10 minutes to promote the full swelling of the gelatin;

[0059] (2) first step of enzymatic hydrolysis: using neutral protease to perform one-step enzymatic hydrolysis on the dissolved fish skin gelatin, the enzyme addition amount is 6000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis time is 2.5h;

[0060] (3) Second step of enzymatic hydrolysis: After the enzymatic hydrolysis by neutral protease, the fish skin gelatin hydrolysate was subjected to secondary enzymatic hydrolysis using an appropriate amount of papain, the enzyme addition amount was 2500 U / g, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 1.5 h;

[0061] (4) Ultrafiltration membrane filtration: The fish skin gelatin hydrolyzate after two-step enzymatic hydrolysis is subjected to ultrafiltration membrane filtration to remove the high molecular weight peptides with a molecular weight>1 kDa that are not fully hydrolyzed. The membrane cutoff molecular weight used is 2 kDa, the membrane concentration multiple is 4 times, and the effluent is collected;

[0062] (5) First step: Nanofiltration membrane filtration: The collected ultrafiltration effluent mainly contains oligopeptides with a molecular weight of <2 kDa. The oligopeptide mixture is further separated using a nanofiltration membrane. A nanofiltration membrane with a molecular weight cutoff of 1000 Da is selected to separate the tripeptides, with a concentration factor of 4. The tripeptides are mostly distributed in the effluent;

[0063] (6) Second step: Nanofiltration: The effluent collected in the first step of nanofiltration is mainly tripeptides, free amino acids and salts. The purpose of this step is to remove the free amino acids and salts mixed in the tripeptides. A nanofiltration membrane with a molecular weight cutoff of 260 Da is selected for treatment, the concentration multiple is 4 times, and then 2 times of water is added to further wash out the free amino acids and salts;

[0064] (7) Low-temperature pasteurization: low-temperature pasteurization of the concentrated solution obtained in step (6);

[0065] (8) Freeze-drying: freeze-dry the sterilized fish skin collagen tripeptide concentrate obtained in step (7) to obtain a high-purity fish skin collagen tripeptide powder.

[0066] In this embodiment, the final yield of high-purity tripeptide was measured to be 69.3%, of which the purity of the tripeptide was 84.8%, the hydrolysis rate was 32.5%, the product powder was fine, the saltiness was moderate, and there was no obvious bitter smell.

[0067] Embodiment 3:

[0068] (1) Fish skin gelatin swelling: The fish skin gelatin was prepared into a solution with a mass fraction of 8%, and heated at 90° C. for 10 minutes to promote the full swelling of the gelatin;

[0069] (2) First step of enzymatic hydrolysis: using neutral protease to perform one-step enzymatic hydrolysis on the dissolved fish skin gelatin, the enzyme addition amount is 8000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis time is 2.5h;

[0070] (3) Second step of enzymatic hydrolysis: After the enzymatic hydrolysis by neutral protease, the fish skin gelatin hydrolysate was subjected to secondary enzymatic hydrolysis using an appropriate amount of papain, the enzyme addition amount was 2500 U / g, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 1.5 h;

[0071] (4) Ultrafiltration membrane filtration: The fish skin gelatin hydrolyzate after two-step enzymatic hydrolysis is subjected to ultrafiltration membrane filtration to remove the high molecular weight peptides with a molecular weight>1 kDa that are not fully hydrolyzed. The membrane cutoff molecular weight used is 2 kDa, the membrane concentration multiple is 4 times, and the effluent is collected;

[0072] (5) First step: Nanofiltration membrane filtration: The collected ultrafiltration effluent mainly contains oligopeptides with a molecular weight of <2 kDa. The oligopeptide mixture is further separated using a nanofiltration membrane. A nanofiltration membrane with a molecular weight cutoff of 1000 Da is selected to separate the tripeptides, with a concentration factor of 4. The tripeptides are mostly distributed in the effluent;

[0073] (6) Second step: Nanofiltration: The effluent collected in the first step of nanofiltration is mainly tripeptides, free amino acids and salts. The purpose of this step is to remove the free amino acids and salts mixed in the tripeptides. A nanofiltration membrane with a molecular weight cutoff of 260 Da is selected for treatment, the concentration multiple is 4 times, and then 2 times of water is added to further wash out the free amino acids and salts;

[0074] (7) Low-temperature pasteurization: low-temperature pasteurization of the concentrated solution obtained in step (6);

[0075] (8) Freeze-drying: freeze-dry the sterilized fish skin collagen tripeptide concentrate obtained in step (7) to obtain a high-purity fish skin collagen tripeptide powder.

[0076] In this embodiment, the final yield of high-purity tripeptide was measured to be 67.9%, of which the purity of the tripeptide was 84.2%, and the hydrolysis rate was 33.9%. The product powder was fine, moderately salty, and had no obvious bitter smell.

[0077] Embodiment 4:

[0078] (1) Fish skin gelatin swelling: The fish skin gelatin was prepared into a solution with a mass fraction of 8%, and heated at 90° C. for 10 minutes to promote the full swelling of the gelatin;

[0079] (2) First step of enzymatic hydrolysis: using neutral protease to perform one-step enzymatic hydrolysis on the dissolved fish skin gelatin, the enzyme addition amount is 4000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis time is 2.5h;

[0080] (3) Second step of enzymatic hydrolysis: After the enzymatic hydrolysis by neutral protease, the fish skin gelatin hydrolysate was subjected to secondary enzymatic hydrolysis using an appropriate amount of papain, the enzyme addition amount was 2500 U / g, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 1.5 h;

[0081] (4) Ultrafiltration membrane filtration: The fish skin gelatin hydrolyzate after two-step enzymatic hydrolysis is subjected to ultrafiltration membrane filtration to remove the high molecular weight peptides with a molecular weight>1 kDa that are not fully hydrolyzed. The membrane cutoff molecular weight used is 2 kDa, the membrane concentration multiple is 4 times, and the effluent is collected;

[0082] (5) First step: Nanofiltration membrane filtration: The collected ultrafiltration effluent mainly contains oligopeptides with a molecular weight of <2 kDa. The oligopeptide mixture is further separated using a nanofiltration membrane. A nanofiltration membrane with a molecular weight cutoff of 1000 Da is selected to separate the tripeptides, with a concentration factor of 4. The tripeptides are mostly distributed in the effluent;

[0083] (6) Second step: Nanofiltration: The effluent collected in the first step of nanofiltration is mainly tripeptides, free amino acids and salts. The purpose of this step is to remove the free amino acids and salts mixed in the tripeptides. A nanofiltration membrane with a molecular weight cutoff of 260 Da is selected for treatment, the concentration multiple is 4 times, and then 2 times of water is added to further wash out the free amino acids and salts;

[0084] (7) Low-temperature pasteurization: low-temperature pasteurization of the concentrated solution obtained in step (6);

[0085] (8) Freeze-drying: freeze-dry the sterilized fish skin collagen tripeptide concentrate obtained in step (7) to obtain a high-purity fish skin collagen tripeptide powder.

[0086] In this embodiment, the final yield of high-purity tripeptide was measured to be 35.6%, of which the purity of the tripeptide was 83.6%, and the hydrolysis rate was 19.1%. The product powder was fine, moderately salty, and had no obvious bitter smell.

[0087] Embodiment 5:

[0088] (1) Fish skin gelatin swelling: The fish skin gelatin was prepared into a solution with a mass fraction of 8%, and heated at 90° C. for 10 minutes to promote the full swelling of the gelatin;

[0089] (2) First step of enzymatic hydrolysis: using neutral protease to perform one-step enzymatic hydrolysis on the dissolved fish skin gelatin, the enzyme addition amount is 9000U / g, the enzymatic hydrolysis temperature is 55°C, and the enzymatic hydrolysis time is 2.5h;

[0090] (3) Second step of enzymatic hydrolysis: After the enzymatic hydrolysis by neutral protease, the fish skin gelatin hydrolysate was subjected to secondary enzymatic hydrolysis using an appropriate amount of papain, the enzyme addition amount was 2500 U / g, the enzymatic hydrolysis temperature was 55° C., and the enzymatic hydrolysis time was 1.5 h;

[0091] (4) Ultrafiltration membrane filtration: The fish skin gelatin hydrolyzate after two-step enzymatic hydrolysis is subjected to ultrafiltration membrane filtration to remove the high molecular weight peptides with a molecular weight>1 kDa that are not fully hydrolyzed. The membrane cutoff molecular weight used is 2 kDa, the membrane concentration multiple is 4 times, and the effluent is collected;

[0092] (5) First step: Nanofiltration membrane filtration: The collected ultrafiltration effluent mainly contains oligopeptides with a molecular weight of <2 kDa. The oligopeptide mixture is further separated using a nanofiltration membrane. A nanofiltration membrane with a molecular weight cutoff of 1000 Da is selected to separate the tripeptides, with a concentration factor of 4. The tripeptides are mostly distributed in the effluent;

[0093] (6) Second step: Nanofiltration: The effluent collected in the first step of nanofiltration is mainly tripeptides, free amino acids and salts. The purpose of this step is to remove the free amino acids and salts mixed in the tripeptides. A nanofiltration membrane with a molecular weight cutoff of 260 Da is selected for treatment, the concentration multiple is 4 times, and then 2 times of water is added to further wash out the free amino acids and salts;

[0094] (7) Low-temperature pasteurization: low-temperature pasteurization of the concentrated solution obtained in step (6);

[0095] (8) Freeze-drying: freeze-dry the sterilized fish skin collagen tripeptide concentrate obtained in step (7) to obtain a high-purity fish skin collagen tripeptide powder.

[0096] In this embodiment, the final yield of high-purity tripeptide was measured to be 61.5%, of which the purity of the tripeptide was 78.6%, and the hydrolysis rate was 34.2%. The product powder was fine, moderately salty, and had no obvious bitter smell.

[0097] From the results obtained in Examples 1-5, it can be seen that the amount of neutral protease added is a key factor affecting the enzymatic efficiency and peptide yield. The amount of enzyme added is controlled at 5000-8000 U / g to ensure that the hydrolysis rate is maintained at 30%-35%. The amount of enzyme added below this range will lead to a significant decrease in the hydrolysis rate and the purity of the tripeptide; the amount of enzyme added above this range will not further increase the hydrolysis rate, but will lead to a decrease in the purity of the tripeptide, because after too much enzyme is added, the hydrolysis limit is almost reached, and some enzymes act on the hydrolysis of small molecule peptides, decomposing them into dipeptides with lower molecular weights, which are difficult to separate from the tripeptide in the ultrafiltration-nanofiltration stage, so the purity of the tripeptide is finally reduced.

[0098] Comparative Example 1:

[0099] The type of enzyme used in the first step of enzymatic hydrolysis in step (2) of Example 1 was changed (alkaline protease, papain and pepsin were added for comparison) and the other conditions were the same as those in Example 1. The effects of the type of enzyme on the hydrolysis rate, tripeptide yield and tripeptide purity are shown in Table 1.

[0100] Table 1 Effect of enzyme type on hydrolysis rate, tripeptide yield and tripeptide purity in comparative example 1

[0101]

[0102] From the results corresponding to Table 1 and Examples 1-5, it can be seen that the use of neutral protease in the first enzymatic hydrolysis process can improve the hydrolysis rate, tripeptide yield and tripeptide purity. The hydrolysis rates of fish skin collagen by alkaline protease, papain and pepsin are significantly lower than those by neutral protease, and alkaline protease and pepsin may produce more dipeptides and tetrapeptides with molecular weights close to tripeptides after hydrolysis, which are difficult to completely separate from the tripeptides, so the tripeptide purity in the obtained product is significantly reduced.

[0103] Comparative Example 2:

[0104] Step (5) in Example 1 was deleted, and other conditions were the same as those in Example 1. The hydrolysis rate, tripeptide yield and tripeptide purity were measured, and the specific results are shown in Table 2.

[0105] Comparative Example 3:

[0106] Step (2) and step (3) in Example 1 were changed into a one-step enzymatic hydrolysis, and 5000U / g neutral protease and 2500U / g papain were added to the fish skin gelatin solution obtained in step (1) at the same time. The enzymatic hydrolysis temperature was 55°C and the enzymatic hydrolysis time was 2.5h. The hydrolysis rate, tripeptide yield and tripeptide purity were measured, and the specific results are shown in Table 2.

[0107] Comparative Example 4:

[0108] The implementation order of step (2) and step (3) in Example 1 was exchanged, and the fish skin gelatin solution was first enzymolyzed using 2500U / g papain, the enzymolysis temperature was 55°C, and the enzymolysis time was 1.5h; then the dissolved fish skin gelatin was subjected to secondary enzymolysis using neutral protease, the enzyme addition amount was 5000, the enzymolysis temperature was 55°C, and the enzymolysis time was 2.5h. The hydrolysis rate, tripeptide yield and tripeptide purity were measured, and the specific results are shown in Table 2.

[0109] Table 2 Hydrolysis rate, tripeptide yield and tripeptide purity corresponding to comparative examples 2-4

[0110]

[0111] As can be seen from Table 2, in Comparative Example 2, the first nanofiltration step of step (5) of Example 1 is removed, and the yield of the obtained collagen peptide product is improved, but the purity of the tripeptide in the product is greatly reduced because the oligopeptides with a molecular weight of 500Da < <2000Da cannot be effectively separated.

[0112] In Comparative Example 3, the step-by-step enzymatic hydrolysis of steps (2) and (3) in Example 1 was adjusted to a single enzymatic hydrolysis by adding enzymes simultaneously. In Comparative Example 4, the order of steps (2) and (3) in Example 1 was adjusted. The results both showed a sharp decrease in the peptide yield because the synergistic effect of the two enzymes could not be fully exerted. This fully demonstrates that the use of neutral protease to fully hydrolyze fish skin collagen is beneficial to the exposure of collagenase sites, thereby maximizing the collagenase hydrolysis efficiency.

[0113] 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 high-purity fish skin collagen tripeptide, characterized in that: The preparation method comprises the following steps: (1) Fish skin gelatin swelling; (2) using a neutral protease to enzymolyze the swollen fish skin gelatin to obtain a fish skin gelatin hydrolyzate; the amount of the neutral protease added is 5000-8000 U / g; the enzymolysis temperature is 50-65° C., and the enzymolysis time is 2-3 h; (3) using papain to enzymolyze the fish skin gelatin hydrolyzate obtained in step (2) to obtain a fish skin gelatin hydrolyzate; the amount of papain added is 2000-3000 U / g; the enzymolysis temperature is 50-65° C., and the enzymolysis time is 1.5-2 h; (4) filtering the fish skin gelatin hydrolyzate obtained in step (3) using an ultrafiltration membrane, and collecting the effluent to remove the high molecular weight peptides with a molecular weight of >1 kDa that are not fully hydrolyzed; (5) filtering the effluent obtained in step (4) by the first step of nanofiltration, collecting the effluent, and separating the fish skin collagen tripeptide; the nanofiltration membrane of the first step of nanofiltration has a molecular weight cutoff of 800-1000Da, and a concentration multiple of 4-5 times; (6) filtering the effluent obtained in step (5) by a second nanofiltration step to remove free amino acids and salts mixed in the fish skin collagen tripeptide; the nanofiltration membrane of the second nanofiltration step has a molecular weight cutoff of 260Da, and a concentration multiple of 3-4 times, and after concentration, 1-2 times the volume of water is added to wash out the free amino acids and salts; (7) Low temperature pasteurization; (8) Freeze drying.

2. The preparation method according to claim 1, characterized in that: The swelling in step (1) is to prepare the fish skin gelatin into a solution with a mass fraction of 5%-10%, and heat it at 80-100° C. for 10 minutes.

3. The preparation method according to claim 1, characterized in that: The ultrafiltration membrane in step (4) has a molecular weight cut-off of 1-2 kDa and a membrane concentration multiple of 4-5 times.

4. A fish skin collagen tripeptide obtained by the preparation method according to any one of claims 1 to 3.

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