A method for preparing fish bone active polypeptide by enzymolysis using immobilized enzyme

Abstract

The invention discloses a method for preparing fish bone active polypeptide by enzymolysis with immobilized enzyme, and relates to a method for extracting polypeptide by enzymolysis. The method comprises the following steps: weighing fresh fish bones, pouring liquid nitrogen and then crushing into particles; mixing the particles and water at a weight ratio of 1:10, steaming at 120°C for 20-30min, removing upper fat, and beating; adding immobilized enzyme into the slurry for enzymolysis, wherein the enzymolysis conditions are as follows: pH 7.5-8, temperature 40-50°C, immobilized enzyme addition amount of 0.8-1mg / mL, time 3-4h, inactivating the enzyme in a boiling water bath, removing upper fat from the obtained enzymolysis solution, vacuum concentrating the separated supernatant to obtain active polypeptide solution, and washing and drying the separated precipitate to obtain high calcium fish bone powder; enzymolysis is performed by using the immobilized enzyme, and the prepared immobilized enzyme has a relatively large enzyme loading capacity, a high enzyme activity yield, stable performance, and can be reused.

Description

Technical Field

[0001] The invention relates to a method for extracting polypeptides by enzymatic hydrolysis, and in particular to a method for preparing fish bone active polypeptides by enzymatic hydrolysis with an immobilized enzyme. Background Art

[0002] my country has abundant fish resources, and there are many scraps produced during the processing of fish, such as fish scales, fish bones, and internal organs. Fish bones account for a large proportion of them. Their main components are protein, fat, water and ash, and they also contain a large number of trace elements such as calcium and phosphorus. Therefore, fish bones have a prospect of comprehensive utilization. At present, the main utilization methods are: First, they are processed into fish bone powder. The main raw materials for making fish bone powder are fish bones, fish heads, fish skins and other scraps. After deodorization, deodorization, drying, and crushing, they can be used as feed or human supplements; second, they are processed into polypeptide preparations. After the protein on the fish bones is enzymatically hydrolyzed by protease, the active polypeptides formed have multiple functions such as anti-oxidation, lowering blood pressure, preventing osteoporosis, antibacterial, and lowering blood sugar. They are widely used in food, health products, biological hospitals and other fields.

[0003] At present, the process of preparing peptides from fish bones generally uses compound proteases for enzymatic hydrolysis, which requires a large amount of enzymes and has high processing costs. In the current existing technology, immobilized enzymes are used to process fish waste materials. Immobilized enzymes are loaded on a matrix by a certain method to form an enzyme-matrix complex, which is not only highly stable, but also can be recycled repeatedly, greatly saving the cost of enzymatic hydrolysis. However, the loading capacity and reusability of immobilized enzymes in the current existing technology are limited, and there is room for further improvement. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing fish bone active peptides by enzymatic hydrolysis using immobilized enzymes to address the deficiencies in the prior art. The immobilized enzyme is used for enzymatic hydrolysis, and the prepared immobilized enzyme has a relatively large enzyme loading capacity, a high enzyme activity yield, and stable performance, can be reused, and can reduce the production cost of enzymatic hydrolysis and extraction of active peptides.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing fish bone active polypeptides by enzymolysis with an immobilized enzyme, comprising the following steps:

[0007] (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles;

[0008] (2) Mix the granules and water in a weight ratio of 1:10, cook at 120°C for 20-30 min, remove the upper fat layer, and beat;

[0009] (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 7.5-8, temperature 40-50°C, the amount of immobilized enzyme added is 0.8-1 mg / mL, the time is 3-4 hours, the enzyme is inactivated in a boiling water bath, the upper fat layer of the obtained enzymolysis solution is removed, the separated supernatant is vacuum concentrated to obtain the active polypeptide liquid, and the separated precipitate is washed and dried to obtain the high calcium fish bone powder;

[0010] The immobilized enzyme is prepared by the following steps:

[0011] 1) Disperse starch and montmorillonite in water, disperse them uniformly by ultrasonication at 90-100°C, add sodium alginate and sodium hydroxymethyl cellulose solution and stir evenly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1-1.5:2:1:100;

[0012] 2) The mixed solution was dropped into a CaCl2 solution with a mass fraction of 2-4%, and the cross-linking reaction was carried out for 1 hour to obtain gel spheres with a diameter of 1-2 mm, which were frozen for 10-12 hours, and the carrier was obtained after washing and drying;

[0013] 3) Disperse the carrier in a 0.1-0.15 g / mL protease solution at a weight ratio of 1:9-11, adsorb at 20-25°C with shaking for 16-20 hours, wash and dry to obtain the immobilized enzyme.

[0014] Furthermore, the carrier prepared in step 2) is immersed in a pH 5.4 buffer solution, 1-2% of the composite amylase in starch amount is added, stirred and reacted at 50° C. for 10 hours, filtered, washed and dried to obtain a porous carrier.

[0015] Furthermore, the composite amylase is a mixture of α-amylase and amyloglucosidase in a weight ratio of 3-6:1.

[0016] Furthermore, the protease in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1.

[0017] Furthermore, the carrier in step 3) is activated before being dispersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1-2 mg / mL at a solid-liquid ratio of 1:5-8, stirring for 2-2.5 hours, and washing and drying.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present application discloses a method for preparing active peptides from fish bones by enzymatic hydrolysis using immobilized enzymes. In order to reduce the cost of extracting active peptides from fish bones, immobilized enzymes are used for enzymatic hydrolysis. The prepared immobilized enzyme has a relatively large enzyme loading capacity, a high enzyme activity yield, and stable performance, and can be reused.

[0020] 2. The immobilized enzyme in this application uses a gel formed by cross-linking sodium alginate and calcium ions as a skeleton. The starch and sodium carboxymethyl cellulose added can interpenetrate with sodium alginate in the sodium alginate skeleton through hydrogen bonds, thereby increasing the structural strength and stability of the gel. In addition, the -CH2OCH2COO - The spatial volume effect can increase the internal voids of the gel and increase the adsorption performance. In addition, a small amount of montmorillonite is added. Montmorillonite has a large specific surface area and excellent adsorption performance. The silicon oxygen bond on the montmorillonite can form a hydrogen bond with the polar group on the sodium alginate, thereby enhancing the adsorption performance of the gel material and increasing the carrier's loading capacity for proteases.

[0021] 3. After the gel spheres are formed, the present application uses amylase for enzymatic hydrolysis, which can hydrolyze the amorphous regions of starch, thereby forming a microporous structure on the surface and inside of the carrier, thereby increasing its fixation and adsorption properties for proteases.

[0022] 4. In order to further increase the adsorption performance of the carrier of the present application for proteases, the present application activates the carrier before fixing the proteases. Dopamine hydrochloride is used. In a weakly alkaline environment, it can be polymerized and modified on the surface of the carrier and the surface of the voids to increase the surface active groups of the carrier, thereby increasing the loading capacity and stability of the carrier for proteases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The bar graph is a bar graph showing the reproducibility of the immobilized enzyme of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0025] To calculate the enzymolysis efficiency, each example corresponds to a control group, in which the immobilized enzyme is replaced with the corresponding unimmobilized free enzyme. After preparing the immobilized enzyme in each example, the enzyme loading of the immobilized enzyme is detected, and the amount of free enzyme used in the control group is the total enzyme loading on the immobilized enzyme added in the corresponding example.

[0026] Example 1

[0027] A method for preparing fish bone active polypeptides by enzymolysis with an immobilized enzyme, comprising the following steps:

[0028] (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles;

[0029] (2) After mixing the granules and water in a weight ratio of 1:10, steam at 120°C for 20 min, remove the upper fat layer, and beat;

[0030] (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 7.5, temperature 40°C, immobilized enzyme addition amount 0.8 mg / mL, time 3 h, after inactivating the enzyme in a boiling water bath, removing the upper fat layer of the obtained enzymolysis solution, and concentrating the separated supernatant by vacuum to obtain the active polypeptide solution, and washing and drying the separated precipitate to obtain high calcium fish bone powder;

[0031] The immobilized enzyme is prepared by the following steps:

[0032] 1) Dispersing starch and montmorillonite in water, uniformly dispersing by ultrasonic at 90°C, adding sodium alginate and sodium hydroxymethyl cellulose solution and stirring evenly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1:2:1:100;

[0033] 2) The mixed solution was dripped into a 2% CaCl2 solution by column drop method, and the cross-linking reaction was carried out for 1 hour to obtain gel spheres with a diameter of 1-2 mm, which were frozen at -5°C for 12 hours, and the carrier was obtained after washing and drying;

[0034] 3) The carrier was dispersed in a 0.1 g / mL protease solution at a weight ratio of 1:9, adsorbed by shaking at 20°C for 20 h, and then washed and dried to obtain the immobilized enzyme.

[0035] The carrier prepared in step 2) is dispersed in a pH 5.4 buffer solution at a weight ratio of 1:5, and a composite amylase of 1% by weight of the starch in step 1) is added, and the mixture is stirred and reacted at 50° C. for 10 hours. The mixture is filtered, washed, and dried to obtain a porous carrier; the composite amylase is a mixture of α-amylase (50 U / mg) and amyloglucosidase (100 U / mL) at a weight ratio of 3:1.

[0036] The protease in the protease solution in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1.

[0037] The carrier in step 3) is activated before being dispersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1 mg / mL at a solid-liquid ratio of 1:5, stirring for 2 hours, washing and drying.

[0038] Example 2

[0039] A method for preparing fish bone active polypeptides by enzymolysis with an immobilized enzyme, comprising the following steps:

[0040] (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles;

[0041] (2) After mixing the granules and water in a weight ratio of 1:10, steam at 120°C for 25 min, remove the upper fat layer, and beat;

[0042] (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 7.5, temperature 45°C, immobilized enzyme addition amount 0.9 mg / mL, time 3.5 h, after inactivating the enzyme in a boiling water bath, removing the upper fat layer of the obtained enzymolysis solution, and concentrating the separated supernatant by vacuum to obtain the active polypeptide solution, and washing and drying the separated precipitate to obtain high calcium fish bone powder;

[0043] The immobilized enzyme is prepared by the following steps:

[0044] 1) Dispersing starch and montmorillonite in water, uniformly dispersing by ultrasonic at 95°C, adding sodium alginate and sodium hydroxymethyl cellulose solution and stirring evenly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1.2:2:1:100;

[0045] 2) The mixed solution was dripped into a 3% CaCl2 solution by column drop method, and the cross-linking reaction was carried out for 1 hour to obtain gel spheres with a diameter of 1-2 mm, which were frozen at -10°C for 12 hours, and the carrier was obtained after washing and drying;

[0046] 3) The carrier was dispersed in a 0.12 g / mL protease solution at a weight ratio of 1:10, adsorbed with shaking at 25°C for 20 h, and then washed and dried to obtain the immobilized enzyme.

[0047] The carrier prepared in step 2) is immersed in a pH 5.4 buffer solution, and a composite amylase with a starch content of 1.5% is added. The mixture is stirred and reacted at 50° C. for 10 hours. The mixture is filtered, washed, and dried to obtain a porous carrier. The composite amylase is a mixture of α-amylase (50 U / mg) and amyloglucosidase (100 U / mL) in a weight ratio of 4:1.

[0048] The protease solution in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1.

[0049] The carrier in step 3) is activated before being immersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1.5 mg / mL at a solid-liquid ratio of 1:6, stirring for 2.5 hours, washing and drying.

[0050] Example 3

[0051] A method for preparing fish bone active polypeptides by enzymolysis with an immobilized enzyme, comprising the following steps:

[0052] (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles;

[0053] (2) After mixing the granules and water in a weight ratio of 1:10, steam at 120°C for 30 min, remove the upper fat layer, and beat;

[0054] (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 8, temperature 50°C, the amount of immobilized enzyme added is 1 mg / mL, the time is 3-4 hours, the enzyme is inactivated in a boiling water bath, the upper fat layer of the obtained enzymolysis solution is removed, the separated supernatant is vacuum concentrated to obtain the active polypeptide liquid, and the separated precipitate is washed and dried to obtain the high calcium fish bone powder;

[0055] The immobilized enzyme is prepared by the following steps:

[0056] 1) Dispersing starch and montmorillonite in water, uniformly dispersing by ultrasonication at 100°C, adding sodium alginate and sodium hydroxymethyl cellulose solution and stirring uniformly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1.5:2:1:100;

[0057] 2) The mixed solution was dripped into a 4% CaCl2 solution by column drop method, and the cross-linking reaction was carried out for 1 hour to obtain gel spheres with a diameter of 1-2 mm, which were frozen at -10°C for 10-12 hours, and the carrier was obtained after washing and drying;

[0058] 3) The carrier was dispersed in a 0.15 g / mL protease solution at a weight ratio of 1:11, adsorbed with shaking at 25°C for 20 h, and then washed and dried to obtain the immobilized enzyme.

[0059] The carrier prepared in step 2) is immersed in a pH 5.4 buffer solution, and a composite amylase with a starch content of 2% is added. The mixture is stirred and reacted at 50° C. for 10 hours. The mixture is filtered, washed, and dried to obtain a porous carrier. The composite amylase is a mixture of α-amylase (50 U / mg) and amyloglucosidase (100 U / mL) in a weight ratio of 5:1.

[0060] The protease solution in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1.

[0061] The carrier in step 3) is activated before being immersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1.8 mg / mL at a solid-liquid ratio of 1:5-8, stirring for 2 hours, washing and drying.

[0062] Example 4

[0063] A method for preparing fish bone active polypeptides by enzymolysis with an immobilized enzyme, comprising the following steps:

[0064] (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles;

[0065] (2) After mixing the granules and water in a weight ratio of 1:10, steam at 120°C for 25 min, remove the upper fat layer, and beat;

[0066] (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 8, temperature 45°C, the amount of immobilized enzyme added is 0.9 mg / mL, the time is 3 h, the enzyme is inactivated in a boiling water bath, the upper fat layer of the obtained enzymolysis solution is removed, the separated supernatant is vacuum concentrated to obtain the active polypeptide liquid, and the separated precipitate is washed and dried to obtain high calcium fish bone powder;

[0067] The immobilized enzyme is prepared by the following steps:

[0068] 1) Dispersing starch and montmorillonite in water, uniformly dispersing by ultrasonic at 95°C, adding sodium alginate and sodium hydroxymethyl cellulose solution and stirring evenly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1.5:2:1:100;

[0069] 2) The mixed solution was dripped into a 3% CaCl2 solution by column drop method, and the cross-linking reaction was carried out for 1 hour to obtain gel spheres with a diameter of 1-2 mm, which were frozen at -10°C for 12 hours, and the carrier was obtained after washing and drying;

[0070] 3) The carrier was dispersed in a 0.12 g / mL protease solution at a weight ratio of 1:10, adsorbed with oscillation at 25°C for 18 h, and then washed and dried to obtain the immobilized enzyme.

[0071] The carrier prepared in step 2) is immersed in a pH 5.4 buffer solution, and a composite amylase with a starch content of 2.5% is added. The mixture is stirred and reacted at 50° C. for 10 hours. The mixture is filtered, washed, and dried to obtain a porous carrier. The composite amylase is a mixture of α-amylase (50 U / mg) and amyloglucosidase (100 U / mL) in a weight ratio of 6:1.

[0072] The protease solution in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1.

[0073] The carrier in step 3) is activated before being immersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1.5 mg / mL at a solid-liquid ratio of 1:8, stirring for 2 hours, washing and drying.

[0074] Comparative Example 1

[0075] Comparative Example 1 is a comparative example of Example 4, except that the carrier prepared in step 2) is not enzymatically hydrolyzed by the composite amylase, and the other processes are the same as those of Example 4.

[0076] Comparative Example 2

[0077] Comparative Example 2 is a comparative example of Example 4, except that: the carrier prepared in step 2) is not activated before adsorbing the protease, and the other processes are the same as Example 4.

[0078] Performance Testing

[0079] 1. The polypeptide content of the enzymatically hydrolyzed supernatant in step (3) of Examples 1-4 and Comparative Examples 1-2 was detected, and the polypeptide conversion rate and enzymatic hydrolysis efficiency were calculated, wherein the polypeptide conversion rate (%) = polypeptide content / protein content*100%, and the polypeptide content was determined by the Gly-Gly-Tyr-Arg tetrapeptide standard method; the enzymatic hydrolysis efficiency (%) = (content of polypeptide hydrolyzed by the immobilized enzyme / enzyme activity of the immobilized enzyme) / (content of polypeptide hydrolyzed by the free enzyme / enzyme activity of the free enzyme). The results are shown in Table 1.

[0080] 2. The loading amount (mg / g), enzyme activity (U / mg) and enzyme activity yield (%) of the immobilized enzymes prepared in Examples 1-4 and Comparative Examples 1-2 were detected; wherein loading amount = (added enzyme content - supernatant enzyme content) / carrier content; enzyme activity yield = total activity of immobilized enzyme / total activity of free enzyme * 100%; the results are shown in Table 1.

[0081] 3. Repeated use performance: The immobilized enzyme prepared in Example 4 was subjected to the enzymatic hydrolysis experiment in Example 4 for 6 times, and its relative enzyme activity was determined to investigate its stability in use, wherein relative enzyme activity = enzyme activity under a certain condition / highest enzyme activity under the condition*100%. The results are shown in Figure 1 .

[0082]

[0083] As can be seen from Table 1, the immobilized enzymes prepared in Examples 1-4 of the present application have a loading capacity of 420.25-427.64 mg / g for protease, and an enzyme activity yield of 87-88.6%, which is relatively large for enzyme loading, high enzyme activity yield, and excellent performance. Moreover, taking the free enzymolysis efficiency as 100%, the enzymolysis efficiency of the immobilized enzyme of the present application can reach 96.1-97.6%, which is almost the same as the free enzyme enzymolysis effect, indicating that the enzymolysis performance of the immobilized enzyme prepared in the present application is excellent.

[0084] In Comparative Example 1, no starch enzymatic hydrolysis was used, and its carrier loading capacity was significantly reduced, which resulted in a low enzymatic polypeptide conversion rate; in Comparative Example 2, the carrier was not activated, and its protease loading capacity was much lower than that of Example 4, indicating that the use of amylase for enzymatic hydrolysis in the present application can improve the adsorption performance of the carrier for the enzyme, thereby increasing the enzyme loading capacity and enzyme activity; and activating the carrier can also greatly increase its adsorption performance with the enzyme and increase the activity of the enzyme.

[0085] In addition, combined Figure 1 It can be seen that the immobilized enzyme prepared in the present application can still maintain a relative enzyme activity of 70.1% after 6 repeated uses, indicating that the immobilized enzyme prepared in the present application has stable performance and can be reused many times, thereby reducing the enzymatic hydrolysis cost of fish bone active peptides.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing fish bone active polypeptide by enzymolysis with an immobilized enzyme, characterized in that: The following steps are involved: (1) Weigh fresh fish bones, pour liquid nitrogen on them and crush them into particles; (2) Mix the granules and water in a weight ratio of 1:10, cook at 120°C for 20-30 min, remove the upper fat layer, and beat; (3) adding immobilized enzyme to the slurry for enzymolysis, the enzymolysis conditions are: pH 7.5-8, temperature 40-50°C, the amount of immobilized enzyme added is 0.8-1 mg / mL, the time is 3-4 hours, the enzyme is inactivated in a boiling water bath, the upper fat layer of the obtained enzymolysis solution is removed, the separated supernatant is vacuum concentrated to obtain the active polypeptide liquid, and the separated precipitate is washed and dried to obtain the high calcium fish bone powder; The immobilized enzyme is prepared by the following steps: 1) Disperse starch and montmorillonite in water, disperse them uniformly by ultrasonication at 90-100°C, add sodium alginate and sodium hydroxymethyl cellulose solution and stir evenly, wherein the weight ratio of starch, montmorillonite, sodium alginate, sodium hydroxymethyl cellulose and water is 0.5:1-1.5:2:1:100; 2) The mixed solution was dropped into a CaCl2 solution with a mass fraction of 2-4%, and the cross-linking reaction was carried out for 1 hour to obtain gel balls, which were frozen for 10-12 hours, and the carrier was obtained after washing and drying; 3) Disperse the carrier in a 0.1-0.15 g / mL protease solution at a weight ratio of 1:9-11, adsorb at 20-25°C with shaking for 16-20 hours, wash and dry to obtain the immobilized enzyme; The carrier prepared in step 2) is immersed in a pH 5.4 buffer solution, 1-2% of the composite amylase in starch amount is added, and the mixture is stirred and reacted at 50° C. for 10 hours. After filtering, the mixture is washed and dried to obtain a porous carrier; The composite amylase is a mixture of α-amylase and amyloglucosidase in a weight ratio of 3-6:1; The protease in the protease solution in step 3) is a mixture of alkaline protease and papain in a weight ratio of 1:1; The carrier in step 3) is activated before being dispersed in the protease solution. The specific treatment steps are: immersing the carrier in a phosphate buffer solution of dopamine hydrochloride at pH 8.5 and 1-2 mg / mL at a solid-liquid ratio of 1:5-8, stirring for 2-2.5 hours, washing and drying.

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