A fan shell polypeptide with weight loss function and its preparation method

Abstract

The present invention relates to the technical field of deep processing of scallops, and particularly relates to a scallop polypeptide with weight loss function and a preparation method thereof. The preparation method of this scallop polypeptide contains scallop polypeptide A with inhibitory activity against α-amylase and / or scallop polypeptide B with inhibitory activity against pancreatic lipase; wherein, the scallop polypeptide A is obtained by hydrolyzing scallop slurry with papain and acid protease successively twice, and then through centrifugation to remove residues, microfiltration, ultrafiltration and nanofiltration to obtain a polypeptide solution with a molecular weight cut-off of 200-1000 Da, and then spray-dried into powder; the scallop polypeptide B is obtained by pretreating scallop slurry with aspergillus lipase to obtain a pretreatment solution, and the pretreatment solution is successively hydrolyzed with 3.4310 acid protease and 537 acid protease twice, and then centrifuged to remove residues, filtered, ultrafiltered to obtain a polypeptide solution with a molecular weight cut-off below 1000 Da, and then spray-dried into powder. The scallop polypeptide with weight loss function and the preparation method thereof provided by the present invention can be used in weight loss and lipid-lowering foods or functional products.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of scallops, and particularly relates to a scallop polypeptide with weight loss function and a preparation method thereof. Background Art

[0002] In recent years, the scallop aquaculture industry in China has developed rapidly, with an annual output reaching hundreds of thousands of tons. Existing research reports and patents on scallop polypeptides include: "Isolation, Identification and Hypotensive Activity Study of Scallop ACE Inhibitory Peptides" written by Li Jingjing et al., in which a polypeptide solution obtained by hydrolyzing scallops with papain was proven to have an inhibitory effect on ACE through mouse experiments. Cui Jinhui reported in the article "Study on the Preparation Process and Biological Activity of Hydrolyzed Protein from Patinopecten yessoensis" that the scallop polypeptide solution hydrolyzed by Bacillus subtilis neutral protease and trypsin has a much greater effect on scavenging hydroxyl radicals than VC. "Optimization of Preparation of Scallop Antioxidant Peptide by Response Surface Orthogonal Rotation Method" written by Li Cai et al., in which the hydrolysate obtained by hydrolyzing scallop skirt with neutral protease has antioxidant properties. In "Effect of Temperature on Amino Nitrogen and Determination of Odor Inhibition Process in Scallop Enzymolysis" written by Chi Yusen et al., the polypeptide solution obtained by hydrolyzing scallops with protease has the function of odor inhibition.

[0003] In the patent document with the publication number CN105483196A (publication date: April 13, 2016), a disclosed ACE inhibitory enzyme solution, its preparation method and application, it is recorded that hydrolyzing scallops with α-chymotrypsin, the obtained polypeptide solution has the effect of lowering blood pressure. Another example is the preparation method of scallop edge whitening peptide disclosed in the patent document with the publication number CN107523601B (publication date: October 11, 2019), that is, scallops are enzymolyzed by pepsin and trypsin to obtain a hydrolysate, which is precipitated with ethanol and recovered and purified, and then a bioactive polypeptide with tyrosinase inhibitory activity can be obtained through ultrafiltration with 5000Da.

[0004] In recent years, scientific research has shown that α-amylase inhibitory peptides have the effects of weight loss and lipid-lowering (see "Preparation and Thermal Stability Study of White Kidney Bean Polypeptides with α-Amylase Inhibitory Activity" written by Zi Yan et al.); pancreatic lipase inhibitory peptides have the effect of weight loss (see "Isolation, Purification and Identification of Camel Blood Protein Inhibiting Pancreatic Lipase" written by Sun Ruxin et al.). It has also been found that simultaneous intake of α-amylase inhibitor and pancreatic lipase inhibitor can significantly improve the weight loss effect (see "Preliminary Exploration on the Inhibitory Activity and Mechanism of Cleistocalyx operculatus on Pancreatic Lipase and α-Amylase" written by Zhang Lina). However, there is currently no report on processing scallops to produce polypeptides with inhibitory activities on α-amylase and pancreatic lipase. Therefore, further research and development of scallop foods with weight loss function are very beneficial to promoting the deep processing of scallops. Summary of the Invention

[0005] To solve the problems mentioned in the above background art, the present invention provides a preparation method of polypeptides from scallop with weight loss function, which contains polypeptides from scallop A with inhibitory activity against α-amylase and / or polypeptides from scallop B with inhibitory activity against pancreatic lipase;

[0006] Among them, the polypeptides from scallop A are prepared by hydrolyzing scallop slurry with papain and acid protease successively twice, and then through centrifugation to remove residues, microfiltration, ultrafiltration and nanofiltration to obtain a polypeptide solution with a molecular weight cut-off of 200-1000 Da, and then spray drying into powder;

[0007] The polypeptides from scallop B are prepared by pretreating scallop slurry with aspergillus lipase to obtain a pretreatment solution, and the pretreatment solution is then hydrolyzed with 3.4310 acid protease and 537 acid protease successively twice, and then centrifuged to remove residues, filtered and ultrafiltered to obtain a polypeptide solution with a molecular weight cut-off of less than 1000 Da, and then spray drying into powder.

[0008] In one embodiment, the preparation method of the scallop slurry is to mix scallop and water at a mass ratio of 1:10-15, heat to 80-90 °C, and keep warm and soften for 1-3 hours; then pour it into a colloid mill and grind the slurry in a cycle for 2-3 minutes to form a slurry.

[0009] In one embodiment, in the preparation of the polypeptides from scallop A, calculated according to the protein content in the scallop slurry used, the addition amount of papain is 4000-8000 u / g, and the addition amount of acid protease is 3000-7000 u / g.

[0010] In one embodiment, in the preparation of the polypeptides from scallop A, the hydrolysis conditions of papain are a temperature of 50-65 °C, a pH range of 5.5-7.5, a hydrolysis time of 80-160 min, and boiling to inactivate the enzyme for 5-10 min after hydrolysis;

[0011] The hydrolysis conditions of the acid protease are a temperature of 40-55 °C, a pH range adjusted with acetic acid of 2.5-3.5, a hydrolysis time of 60-120 min, and inactivating the enzyme at 80-90 °C for 3-6 min after hydrolysis.

[0012] In one embodiment, in the preparation of the polypeptides from scallop A, after the scallop hydrolyzate after two enzymatic hydrolyses is centrifuged at 4000-6000 rpm to remove residues, the obtained supernatant is microfiltered through a 0.22-0.45 μm membrane, the microfiltrate is ultrafiltered through a 1000 Da ultrafiltration membrane, and the filtrate is nanofiltrated through a 200 Da nanofiltration membrane and concentrated to a polypeptide content of 5%-7% to obtain a nanofiltration concentrated polypeptide solution with a molecular weight of 200-1000 Da.

[0013] In one embodiment, in the preparation of the fan shell polypeptide A, the polypeptide solution after nanofiltration concentration is also subjected to vacuum concentration and acetic acid is removed before spray drying, so that the pH value reaches 5.0 - 6.5. When the fan shell polypeptide A prepared after spray drying is diluted to a polypeptide content of 0.4% - 1.8%, the inhibition rate against α-amylase reaches 38% - 65%.

[0014] In one embodiment, in the preparation of the fan shell polypeptide B, the addition amount of the Aspergillus lipase is 0.3% - 0.7% of the mass of the scallops used.

[0015] The pretreatment conditions of the Aspergillus pancreatolipase are to adjust the pH to 6.5 - 8.0 with a calcium hydroxide emulsion and treat at a temperature of 40 - 50 °C for 60 - 120 min.

[0016] In one embodiment, in the preparation of the fan shell polypeptide B, calculated according to the protein content in the scallop slurry used, the addition amount of the 3.4310 acid protease is 4000 - 8000 u / g, and the addition amount of the 537 acid protease is 3000 - 7000 u / g.

[0017] In one embodiment, in the preparation of the fan shell polypeptide B, the hydrolysis conditions of the 3.4310 acid protease are to adjust the pH to 2.5 - 3.5 with acetic acid, at a temperature of 40 - 52 °C, and the hydrolysis time is 80 - 160 min. After hydrolysis, the temperature is raised to 80 - 90 °C to inactivate the enzyme for 3 - 5 min;

[0018] The hydrolysis conditions of the 537 acid protease are to adjust the pH to 2.5 - 3.5 with acetic acid, at a temperature of 45 - 58 °C, and the hydrolysis time is 60 - 140 min. After hydrolysis, the enzyme is inactivated at 80 - 90 °C for 3 - 6 min.

[0019] In one embodiment, in the preparation of the fan shell polypeptide B, the scallop hydrolyzate after three enzymatic hydrolyses is first centrifuged at 4000 - 6000 rpm to remove the precipitate residue; the obtained supernatant is filtered with diatomaceous earth, and the filtrate is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight less than 1000 Da.

[0020] In one embodiment, in the preparation of the fan shell polypeptide B, the polypeptide solution ultrafiltered through the ultrafiltration membrane is also subjected to vacuum concentration and acetic acid is removed before spray drying, so that the pH value reaches 5.0 - 6.5. When the fan shell polypeptide B prepared after spray drying is diluted to a polypeptide content of 0.5% - 3.0%, the inhibition rate against pancreatic lipase is 40% - 75%.

[0021] Specifically, the preparation method of the fan shell polypeptide A includes the following preparation steps:

[0022] S100. Mix scallops and water at a mass ratio of 1:10 - 15, heat to 80 - 90 °C, and keep warm and soften for 1 - 3 hours; then pour into a colloid mill and circulate for grinding for 2 - 3 minutes to form a slurry;

[0023] S200. Add 4000 - 8000 u / g of papain according to the protein mass in the raw materials, adjust the pH to 5.5 - 7.5, and carry out the first hydrolysis at 50 - 65 °C for 80 - 160 min. After hydrolysis, boil to inactivate the enzyme for 5 - 10 min;

[0024] S300. Add 3000 - 7000 u / g of acid protease to the first hydrolysis solution after inactivating the enzyme, adjust the pH to 2.5 - 3.5 with acetic acid, and carry out the second hydrolysis at 40 - 55 °C for 60 - 120 min. After the hydrolysis ends, inactivate the enzyme at 80 - 90 °C for 3 - 6 min;

[0025] S400. For the scallop hydrolysis solution after two - step enzymatic hydrolysis, first centrifuge at 4000 - 6000 rpm to remove residues; the supernatant is micro - filtered through a 0.22 - 0.45 μm membrane, the micro - filtrate is ultra - filtered through a 1000 Da ultra - filtration membrane, and the filtrate is nano - filtered through a 200 Da nano - filtration membrane and concentrated to a polypeptide content of 5% - 7% to obtain a nano - filtration concentrated polypeptide solution with a molecular weight of 200 - 1000 Da;

[0026] S500. Vacuum - concentrate the nano - filtration concentrated polypeptide solution and remove acetic acid to make the pH value of the solution reach about 5.0 - 6.5, and then spray - dry it into powder for storage.

[0027] Specifically, the preparation method of the scallop polypeptide B includes the following preparation steps:

[0028] S100. Mix scallops and water at a mass ratio of 1:10 - 15, heat to 80 - 90 °C, and keep warm and soften for 1 - 3 hours; then pour into a colloid mill and circulate for grinding for 2 - 3 minutes to form a slurry;

[0029] S200. Add 0.3% - 0.7% of Aspergillus pancreatin lipase to the scallop slurry according to the scallop mass, adjust the pH to 6.5 - 8.0 with calcium hydroxide emulsion, and carry out pancreatic lipase pretreatment on the scallop slurry; the treatment conditions are a temperature of 40 - 50 °C and a time of 60 - 120 min to obtain a pretreatment solution;

[0030] S300. Calculate according to the protein mass in the raw materials, add 4000 - 8000 u / g of 3.4310 acid protease to the pretreatment solution, adjust the pH to 2.5 - 3.5, and carry out the second hydrolysis at 40 - 52 °C for 80 - 160 min. After hydrolysis, raise the temperature to 80 - 90 °C to inactivate the enzyme for 3 - 5 min;

[0031] S400. Add 3000 - 7000 u / g of 537 acid protease to the second hydrolyzate after enzyme inactivation, adjust the pH to 2.5 - 3.5 with acetic acid, and conduct the third hydrolysis at 45 - 58 °C for 60 - 140 min. After the hydrolysis is completed, inactivate the enzyme at 80 - 90 °C for 3 - 6 min;

[0032] S500. For the scallop hydrolyzate after three enzymatic hydrolyses, first centrifuge at 4000 - 6000 rpm to remove the precipitate residue; filter the supernatant with diatomaceous earth, and then ultrafilter the filtrate with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight less than 1000 Da;

[0033] S600. The polypeptide solution with a molecular weight less than 1000 Da is vacuum - concentrated under an absolute pressure of 0.04 - 0.06 MPa to remove acetic acid, so that the pH value of the solution reaches about 5.0 - 6.5, and then spray - dried into powder for storage.

[0034] The present invention also provides a scallop polypeptide with a weight - loss function, which is prepared by using the preparation method of the scallop polypeptide with a weight - loss function described above.

[0035] Based on the above, compared with the prior art, a scallop polypeptide with a weight - loss function provided by the present invention has the following beneficial effects:

[0036] By hydrolyzing scallops successively with papain and acid protease, the present invention can obtain a polypeptide hydrolyzate with high α - amylase inhibitory activity, while by hydrolyzing scallops successively with 3.4310 acid protease and 537 acid protease, a functional polypeptide with high pancreatic lipase inhibitory activity can be obtained; therefore, after mixing the hydrolyzed polypeptides of the two, it has both inhibitory activities on α - amylase and pancreatic lipase, and is applicable to some lipid - lowering and weight - loss products.

[0037] Other features and beneficial effects of the present invention will be described in the subsequent specification, and part of them will become obvious from the specification or be understood by implementing the present invention. The objectives and other beneficial effects of the present invention can be achieved and obtained through the structures and / or components pointed out in the specification and claims. Detailed embodiments

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0039] In the description of the present invention, it should be noted that all terms used in the present invention (including technical terms and scientific terms) have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention pertains, and should not be construed as limiting the present invention; it should be further understood that the terms used in the present invention should be understood to have meanings consistent with their meanings in the context of this specification and the relevant art, and should not be understood in an idealized or overly formal sense, unless otherwise clearly defined in the present invention.

[0040] Since the fan shell polypeptide with weight loss function provided by the invention contains fan shell polypeptide A with inhibitory activity against α - amylase and / or fan shell polypeptide B with inhibitory activity against pancreatic lipase, therefore, the present invention will provide examples for fan shell polypeptide A and fan shell polypeptide B respectively, so as to better illustrate the respective activity characteristics of fan shell polypeptide A and fan shell polypeptide B.

[0041] The present invention provides the following examples for fan shell polypeptide A

[0042] Example A - 1

[0043] S100. Take 50 g of scallop powder (containing 64% protein), add 500 g of softened water, heat to 85 °C, keep warm for 2 hours, then pour into a colloid mill and circulate for grinding for 2 minutes to form scallop slurry.

[0044] S200. Add 160,000 u of papain according to the protein mass in the slurry, adjust the pH to 6, and hydrolyze at 50 °C for 160 min. After hydrolysis, boil to inactivate the enzyme for 5 min to obtain the first hydrolysis solution;

[0045] S300. Cool the first hydrolysis solution to 45 °C, add 96,000 u of 3.350 acid protease, adjust the pH to 2.5 with acetic acid, and hydrolyze at 45 °C for 120 min. After hydrolysis, inactivate the enzyme at 80 °C for 6 min to obtain the second hydrolysis solution.

[0046] S400. The scallop hydrolysis solution after two - step enzymatic hydrolysis, i.e., the second hydrolysis solution, is centrifuged at 4000 rpm to remove residues; the supernatant is microfiltered through a 0.45 - micron membrane, the filtrate is ultrafiltered through a 1000 - Da ultrafiltration membrane, and the filtrate is concentrated to a polypeptide content of 6% through a 200 - Da nanofiltration membrane to obtain a nanofiltration - concentrated polypeptide solution with a molecular weight of 200 - 1000 Da.

[0047] S500. This nanofiltration - concentrated polypeptide solution is vacuum - concentrated under an absolute pressure of 0.04 MPa to remove acetic acid, and then spray - dried into powder for storage, thus obtaining fan shell polypeptide A.

[0048] For Example A - 1, the present invention also provides the following comparative examples

[0049] Comparative Example A - 1.1

[0050] Replace papain with other enzymes such as bromelain, and the hydrolysis temperature of bromelain is 55°C and the pH is 7.5; other operations and process conditions are the same as those in Example A-1.

[0051] Comparative Example A-1.2

[0052] Replace papain with other enzymes such as ficin, and the hydrolysis temperature of ficin is 60°C and the pH is 7.0; other operations and process conditions are the same as those in Example A-1.

[0053] Comparative Example A-1.3

[0054] Replace papain with other enzymes such as AX protease (produced by Aspergillus oryzae, Amano Enzyme Co., Ltd.), and the hydrolysis conditions of AX protease are 50°C and the pH is 7.0, and other operations and process conditions are the same as those in Example A-1.

[0055] Comparative Example A-1.4

[0056] Replace papain with other enzymes such as subtilisin neutral protease, and the hydrolysis temperature of subtilisin neutral protease is 45°C and the pH is 7.0, and other operations and process conditions are the same as those in Example A-1.

[0057] Comparative Example A-1.5

[0058] Replace papain with other enzymes such as 0.8L neutral protease, and the hydrolysis temperature of 0.8L neutral protease is 47°C and the pH is 7.0, and other operations and process conditions are the same as those in Example A-1.

[0059] Dilute the polypeptide powders prepared in Example A-1 and Comparative Examples A-1.1 to A-1.5 to 15 mg / ml, and measure the α-amylase activity inhibition rate by the 3,5-dinitrosalicylic acid colorimetric method. The specific results are shown in Table 1:

[0060] Table 1

[0061]

[0062] Note: - Not detected

[0063] It can be seen from the detection results in Table 1 that in Comparative Examples A-1.1, A-1.2, and A-1.4 where papain was replaced with different neutral proteases and sulfhydryl proteases for hydrolyzing scallops, no α-amylase inhibitory activity was detected; although the samples in Comparative Examples A-1.3 and A-1.5 had inhibition rates, they were only 7.8% and 15.1% respectively, which were much lower than the inhibition rate of 62.3% in Example 1.

[0064] In addition, the present invention also detected that when the polypeptide content of the polypeptides in scallop polypeptide A prepared in Example A-1 was diluted to 0.5%, its α-amylase inhibition rate was 40.5%. Thus, it can be seen that even at a lower polypeptide content, it still has obvious α-amylase inhibitory activity.

[0065] Example A-2

[0066] S100. Take 100 g of scallop powder (containing 64% protein), add 1500 g of water, heat to 88°C, and keep warm and soften for 2.6 hours; then pour it into a colloid mill and grind the slurry in a cycle for 2.5 minutes. After defoaming, a scallop slurry is formed.

[0067] S200. Add 320,000 u of papain according to the protein mass in the raw materials, adjust the pH to 7.0, and hydrolyze at 65°C for 80 min. After hydrolysis, boil to inactivate the enzyme for 8 min to obtain the first hydrolyzate;

[0068] S300. Cool the first hydrolyzate to 42°C, add 320,000 u of 3.350 acidic protease according to the protein mass, adjust the pH to 3.3 with acetic acid, and hydrolyze at 55°C for 60 min. After hydrolysis, inactivate the enzyme at 82°C for 5 min to obtain the second hydrolyzate.

[0069] S400. The scallop hydrolyzate after two enzymatic hydrolyses, i.e., the second hydrolyzate, is centrifuged at 6000 rpm to remove residues; the supernatant is microfiltered through a 0.22-μm membrane, the microfiltered solution is ultrafiltered through a 1000-Da ultrafiltration membrane, and the filtrate is nanofiltrated and concentrated to a polypeptide content of 5% through a 200-Da nanofiltration membrane to obtain a nanofiltration concentrated polypeptide solution with a molecular weight of 200-1000 Da.

[0070] S500. Vacuum concentrate the nanofiltration concentrated polypeptide solution under an absolute pressure of 0.05 MPa and remove acetic acid, and then spray-dry it into a powder for storage to obtain scallop polypeptide A.

[0071] For Example A-2, the present invention provides the following comparative examples:

[0072] Comparative Example A-2.1

[0073] Use 3.4310 acidic protease (produced by Aspergillus niger 3.4310 strain) with equal enzyme activity to replace 3.350 acidic protease, and the other operations and process conditions are the same as those in Example A-2.

[0074] Comparative Example A-2.2

[0075] Use 537 acidic protease (produced by Aspergillus usamii strain) with equal enzyme activity to replace 3.350 acidic protease, and the other operations and process conditions are the same as those in Example A-2.

[0076] Comparative Example A-2.3

[0077] Replace the 3.350 acid protease with the acid protease produced by Bacillus licheniformis strain, and the hydrolysis conditions of this protease are 40 °C and pH 3.0. Other operations and process conditions are the same as those in Example A-2.

[0078] Dilute the polypeptide powders prepared in Example A-2 and Comparative Examples A-2.1 to A-2.3 to 15 mg / ml, and use the 3,5-dinitrosalicylic acid colorimetric method to test the inhibition rate of α-amylase activity. The specific results are shown in Table 2:

[0079] Table 2

[0080] Example A-2 Comparative Example A-2.1 Comparative Example A-2.2 Comparative Example A-2.3 α-Amylase Activity Inhibition Rate % 62.8％ -- 10.3％ -

[0081] Note: - Not detected

[0082] The comparative experiment found (Table 3) that the hydrolysis solutions of Comparative Examples A-2.1 and A-2.3, which replaced the 3.350 acid protease with 3.4310 acid protease and Bacillus licheniformis acid protease, respectively, had no inhibitory activity on α-amylase. For Comparative Example A-2.2, which replaced the 3.350 acid protease with 537 acid protease, although the hydrolyzed sample had α-amylase inhibitory activity, the activity was low, and the inhibition rate was only 10.3%. A large number of experiments and screenings showed that the polypeptide obtained by hydrolyzing scallops with papain and 3.350 acid protease in sequence had the best inhibitory effect on α-amylase.

[0083] In addition, when the scallop polypeptide A prepared in Example A-2 was diluted to a polypeptide content of 1.7%, the α-amylase inhibition rate of this polypeptide was detected to be 64.2%. When diluted to a polypeptide content of 1.0%, the α-amylase inhibition rate was 52.7%.

[0084] Example A-3

[0085] S100: Take 220 g of scallop powder (containing 64% protein), add 3000 g of softened water, heat to 80 °C, and keep warm for 3 hours; then pour it into a colloid mill and circulate for grinding for 3 minutes to form scallop slurry.

[0086] S200: Add 704,000 u of papain according to the protein mass in the raw materials, adjust the pH to 6.5, and hydrolyze at 55 °C for 120 min. After hydrolysis, boil to inactivate the enzyme for 6 min to obtain the first hydrolysis solution;

[0087] S300: Cool the first hydrolysis solution to 50 °C, add 422,400 u of 3.350 acid protease according to the protein mass, adjust the pH to 3.0 with acetic acid, and hydrolyze at 50 °C for 100 min. After hydrolysis, inactivate the enzyme at 85 °C for 4 min to obtain the second hydrolysis solution;

[0088] S400. The scallop hydrolyzate after secondary enzymatic hydrolysis, i.e., the second hydrolyzate, is centrifuged at 5000 rpm to remove residues; the supernatant is microfiltered through a 0.22-μm membrane, and the microfiltered solution is evacuated of acetic acid under a vacuum at an absolute pressure of 0.06 MPa and appropriately concentrated to adjust the pH to 5.5, obtaining a degassed polypeptide solution.

[0089] S500. The above degassed polypeptide solution is ultrafiltered through a 5000-Da ultrafiltration membrane to obtain a retentate containing polypeptides with a molecular weight >5000 Da and a filtrate with a molecular weight <5000 Da; the polypeptide solution with a molecular weight <5000 Da is ultrafiltered through a 3000-Da ultrafiltration membrane to obtain a filtrate containing polypeptides with a molecular weight <3000 Da and a retentate containing polypeptides with a molecular weight of 5000 - 3000 Da; the polypeptide solution with a molecular weight <3000 Da is further ultrafiltered through a 1000-Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight of 3000 - 1000 Da and a polypeptide solution with a molecular weight <1000 Da. A portion of the polypeptide solution with a molecular weight <1000 Da is nanofiltrated through a 200-Da nanofiltration membrane to obtain a nanofiltrated polypeptide solution with a molecular weight of 200 - 1000 Da. The above polypeptide solutions are each concentrated to a polypeptide content of 1.5%, and the α-amylase activity inhibition rates of different polypeptide solutions are detected, as shown in Table 3.

[0090] Table 3

[0091] Polypeptide Molecular Segment >5000Da 5000 - 3000Da 3000 - 1000Da <1000Da 200 - 1000Da α-Amylase Activity Inhibition Rate % 0 3.2％ 11.6％ 57.9％ 61.3％

[0092] From the test results in Table 3, it can be seen that the active polypeptides with high α-amylase inhibition rates are mainly concentrated in the molecular segment with a molecular weight <1000 Da. Although there is a small amount of inhibitory activity in the molecular segments of 5000 - 3000 Da and 3000 - 1000 Da, it is speculated that this may be due to a small amount of small molecules with a molecular weight <1000 Da not being filtered out. Nanofiltration of the molecular segment with a molecular weight <1000 Da through a 200-Da nanofiltration membrane can improve the α-amylase inhibition activity of the polypeptide solution. Therefore, after the scallops are successively hydrolyzed by papain and 3.350 acidic protease, the polypeptide segment with a molecular weight of 200 - 1000 Da separated is the optimal polypeptide with high α-amylase inhibition activity.

[0093] The present invention provides the following examples regarding scallop polypeptide B

[0094] Example B-1

[0095] S100. Take 100 g of scallop powder (containing 64% protein), add 1000 ml of water to soften it, heat it to 80°C, and keep it warm for 3 hours; then pour it into a colloid mill and circulate for grinding for 3 minutes to form a scallop slurry.

[0096] S200. Add 0.3% of aspergillus lipase (0.3 g) based on the mass of the scallops to the scallop slurry, adjust the pH of the slurry to 6.5 with calcium hydroxide emulsion, and hydrolyze at 40°C for 60 min to obtain a pretreatment solution.

[0097] S300. Add 256,000 U of 3.43×10 acidic protease to the pretreatment solution, adjust the pH to 2.5 with acetic acid, hydrolyze at 40 °C for 80 min, and then raise the temperature to 80 °C to inactivate the enzyme for 5 min to obtain the second hydrolysis solution.

[0098] S400. Cool the second hydrolysis solution to 42 °C, add 192,000 U of 537 acidic protease according to the protein mass in the raw material, adjust the pH to 3 with acetic acid, hydrolyze at 45 °C for 140 min, and then inactivate the enzyme at 80 °C for 6 min to obtain the third hydrolysis solution.

[0099] S500. The scallop hydrolysis slurry after three enzymatic hydrolyses, i.e., the third hydrolysis solution, is centrifuged at 4000 rpm to remove residues; the supernatant is filtered with diatomaceous earth, and the filtrate is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight less than 1000 Da.

[0100] S600. The polypeptide solution is vacuum concentrated under an absolute pressure of 0.04 MPa to remove acetic acid, so that the pH value of the solution reaches 6.2, and then spray-dried into powder for storage to obtain scallop polypeptide B.

[0101] For Example B-1, the present invention also provides the following comparative examples

[0102] Comparative Example B-1.1

[0103] The scallop slurry was not pretreated with aspergillus lipase, and other operations and process conditions were the same as those in Example B-1.

[0104] Comparative Example B-1.2

[0105] The filtrate after diatomaceous earth filtration was nanofiltrated through a 200 Da pore size, and the retentate was ultrafiltered with a 1000 Da ultrafiltration membrane. Other operations and process conditions were the same as those in Example B-1.

[0106] The polypeptide powders prepared in Example B-1, Comparative Example B-1.1, and Comparative Example B-1.2 were diluted to 15 mg / ml, and the inhibition rate of pancreatic lipase was detected by the potentiometric titration method for lipase preparations in GB / T 23535-2009. The detection results are shown in Table 4:

[0107] Table 4

[0108] Example B-1 Comparative Example B-1.1 Comparative Example B-1.2 Pancreatic Lipase Inhibition Rate % 52.0％ 38.6％ 47.8％

[0109] It can be seen from the test results in Table 4 that the inhibition rate of pancreatic lipase in Comparative Example B-1.1 without pretreatment with aspergillus lipase decreased by 25.8% compared with Example B-1, and the inhibition rate decreased significantly; this may be because the fat in scallops that has not been hydrolyzed by aspergillus lipase will hinder the subsequent protease hydrolysis and is not conducive to the generation of active polypeptides.

[0110] The pancreatic lipase inhibition rate of the polypeptide prepared from Comparative Example B-1.2 after nanofiltration treatment decreased by 8.1% compared with that of Example B-1. It may be due to the filtration of a small amount of dipeptides or amino acids with pancreatic lipase inhibitory activity. Therefore, the polypeptide solution cannot be subjected to nanofiltration treatment.

[0111] In addition, when the polypeptide powder prepared in Example B-1 was diluted to a polypeptide content of 0.5%, the measured pancreatic lipase inhibition rate was 40.3%; when diluted to 2.8% of the polypeptide, the measured pancreatic lipase inhibition rate was 74.2%.

[0112] Example B-2

[0113] S100. Take 220 g of scallop powder (containing 64% protein), soften it with 2500 ml of water, heat it to 90 °C, and keep it warm for 2 hours; then pour it into a colloid mill and circulate the grinding for 2 minutes to form scallop slurry.

[0114] S200. Add 0.5% aspergillus lipase (1.1 g) to the scallop slurry according to the mass of the scallop, adjust the pH of the slurry to 7 with calcium hydroxide emulsion, and hydrolyze it at 45 °C for 70 min to obtain a pretreatment solution.

[0115] S300. Add 704,000 U of 3.4310 acidic protease to the pretreatment solution, adjust the pH to 3.5 with acetic acid, hydrolyze it at 52 °C for 90 min, and then raise the temperature to 85 °C to inactivate the enzyme for 4 min to obtain a second hydrolysis solution.

[0116] S400. Cool the second hydrolysis solution to 50 °C, add 423,000 U of 537 acidic protease, adjust the pH to 3.5 with acetic acid, hydrolyze it at 58 °C for 60 min, and after hydrolysis, inactivate the enzyme at 85 °C for 5 min to obtain a third hydrolysis solution.

[0117] S500. The scallop hydrolysis solution after three polypeptide enzymolyses, i.e., the third hydrolysis solution, is centrifuged at 5000 rpm to remove residues; the supernatant is filtered with diatomaceous earth, the pH of the filtrate is adjusted to 6.5, and ultrafiltered with a 5000 Da ultrafiltration membrane to obtain a retentate of polypeptides with a molecular weight > 5000 Da and a filtrate of < 5000 Da; the polypeptide solution of < 5000 Da is ultrafiltered with a 2000 Da ultrafiltration membrane to obtain a polypeptide filtrate of < 2000 Da and a retentate of polypeptides of 5000 - 2000 Da; the polypeptide solution of < 2000 Da is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution of 2000 - 1000 Da and a polypeptide solution of < 1000 Da. Adjust the above different ultrafiltered polypeptides to 15 mg / ml and detect the inhibition rate of pancreatic lipase. The test results are shown in Table 5:

[0118] Table 5

[0119] Polypeptide Molecular Segment >5000Da 2000 - 5000Da 1000 - 2000Da <1000Da Pancreatic Lipase Activity Inhibition Rate % - - 5.3％ 52.0％

[0120] Note: - Not detected

[0121] As can be seen from the test results in Table 5, the polypeptides with high pancreatic lipase inhibitory rate mainly exist in the polypeptide segments below 1000 Da, and the inhibitory rate is 52%. The polypeptide solution with a molecular weight of 1000 - 2000 Da has a slight pancreatic lipase inhibitory rate, only 5.3%, and the pancreatic lipase inhibitory rate of the polypeptide solution with a molecular weight greater than 2000 Da is not detected.

[0122] Example B-3

[0123] S100: Take 500 g of scallop powder (containing 64% protein), soften it with 7000 ml of water, heat it to 85 °C, keep it warm for 2.5 hours, and then pour it into a colloid mill to circulate and grind for 2.5 minutes to form scallop slurry.

[0124] S200: Add 0.7% of Aspergillus pancreatin (3.5 g) to the scallop slurry according to the mass of the scallop, adjust the pH of the slurry to 7.5 with calcium hydroxide emulsion, and hydrolyze it at 50 °C for 120 min to obtain a pretreatment solution.

[0125] S300: After the pretreatment with pancreatin, add 1.6 million U of 3.43×10 acidic protease (produced by Aspergillus niger strain 3.43×10) to the pretreatment solution, adjust the pH to 3 with acetic acid, hydrolyze it at 45 °C for 110 min, and then raise the temperature to 88 °C to inactivate the enzyme for 4.5 min to obtain a second hydrolysis solution.

[0126] S400: Cool the second hydrolysis solution to 48 °C, add 1.28 million U of 537 acidic protease (produced by Aspergillus usamii strain), adjust the pH to 2.5 with acetic acid, hydrolyze it at 50 °C for 100 min, and after hydrolysis, inactivate the enzyme at 86 °C for 5.5 min to obtain a third hydrolysis solution.

[0127] S500: The scallop hydrolysis solution after three polypeptide enzymolyses, that is, the third hydrolysis solution, is centrifuged at 6000 rpm to remove residues; the supernatant is filtered with diatomaceous earth, and the filtrate is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight less than 1000 Da.

[0128] S600: The polypeptide solution is vacuum concentrated under an absolute pressure of 0.06 MPa and acetic acid is removed to make the pH value of the solution reach 6.5, and then it is spray-dried into powder for storage, thus obtaining scallop polypeptide B.

[0129] For Example B-3, the present invention also provides the following comparative examples

[0130] Comparative Example B-3.1

[0131] Replace the enzyme activity of 3.4310 acid protease (produced by Aspergillus niger strain 3.4310) with 3.350 acid protease (produced by Aspergillus niger strain 3.350), and other operations and processes are the same as those in Example B-3.

[0132] Comparative Example B-3.2

[0133] Replace the enzyme activity of 3.4310 acid protease (produced by Aspergillus niger strain 3.4310) with Bacillus licheniformis acid protease (produced by Bacillus licheniformis strain cc5), and the hydrolysis conditions of this protease are 45 °C and pH 3.0. Other operations and processes are the same as those in Example B-3.

[0134] Comparative Example B-3.3

[0135] Replace the enzyme activity of 537 acid protease (produced by Aspergillus usamii strain) with 3.350 acid protease, and other operations and processes are the same as those in Example B-3.

[0136] Comparative Example B-3.4

[0137] Replace the enzyme activity of 537 acid protease (produced by Aspergillus usamii strain) with Bacillus licheniformis acid protease (produced by Bacillus licheniformis strain cc5), and other operations and processes are the same as those in Example B-3.

[0138] Dilute the polypeptide powders prepared in Example B-3, Comparative Example B-3.1, and Comparative Example B-3.4 to 15 mg / ml, and use the potentiometric titration method for lipase preparations in GB / T 23535-2009 to detect the inhibition rate of pancreatic lipase. The test results are shown in Table 6:

[0139] Table 6

[0140] Example B-3 Comparative Example B-3.1 Comparative Example B-3.2 Comparative Example B-3.3 Comparative Example B-3.4 Pancreatic Lipase Inhibition Rate % 52.3％ 8.6％ - - 5.2％

[0141] Note: - Not detected

[0142] It can be seen from the test results in Table 6 that when the 3.4310 acid protease used in the first hydrolysis is replaced with 3.350 acid protease and Bacillus licheniformis acid protease, the inhibition rates of pancreatic lipase in Comparative Example B-3.1 and Comparative Example B-3.2 are 8.6% and not detected, respectively; when the 537 acid protease used in the second hydrolysis is replaced with 3.350 acid protease and Bacillus licheniformis acid protease, the inhibition rates of pancreatic lipase in Comparative Example B-3.3 and Comparative Example B-3.4 are not detected and 5.2%, respectively, which are much lower than 52.3% in Example B-3.

[0143] Thus, it can be seen that the combination of using 3.4310 acid protease for the first hydrolysis and 537 acid protease for the second hydrolysis of scallops is the best enzymatic hydrolysis combination to obtain a polypeptide sample with a high inhibition rate of pancreatic lipase.

[0144] It can be seen from the examples of polypeptides from Chlamys farreri A and polypeptides from Chlamys farreri B that the two respectively have relatively high α-amylase inhibitory activity and pancreatic lipase inhibitory activity. Therefore, the polypeptides from Chlamys farreri provided by the present invention contain polypeptides from Chlamys farreri A with α-amylase inhibitory activity and / or polypeptides from Chlamys farreri B with pancreatic lipase inhibitory activity, and have a certain weight loss function, and are applicable to some products for reducing lipid and losing weight.

[0145] Regarding the proportion relationship between polypeptides from Chlamys farreri A and polypeptides from Chlamys farreri B in the polypeptides from Chlamys farreri provided by the present invention, those skilled in the art can fully make adaptive adjustments according to the different dietary structures targeted by the actual products and the people with relevant needs for weight loss, slimming or weight control. For example, for some people with a relatively high starch intake in their daily diet, the addition proportion of polypeptides from Chlamys farreri A can be appropriately increased, while for some people with a relatively high fat intake in their daily diet, the proportion of polypeptides from Chlamys farreri B can be appropriately increased, that is, it depends on the actual application situation specifically, and will not be elaborated here.

[0146] In addition, those skilled in the art should understand that although there are many problems in the prior art, each embodiment or technical solution of the present invention can be improved only in one or several aspects, and it is not necessary to solve all the technical problems listed in the prior art or the background art at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be regarded as a limitation to that claim.

[0147] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of fan shell polypeptide with weight loss function, characterized in that: It contains polypeptides from scallops A with inhibitory activity against α-amylase and / or polypeptides from scallops B with inhibitory activity against pancreatic lipase; Among them, the polypeptides from scallops A are prepared by subjecting scallop slurry to two hydrolyses with papain and acid protease in sequence, followed by centrifugation to remove residues, microfiltration, ultrafiltration, and nanofiltration to obtain a polypeptide solution with a molecular weight cut-off of 200 - 1000 Da, and then spray-drying into powder; The hydrolysis conditions of the papain are a temperature of 50 - 65 °C, a pH range of 5.5 - 7.5, a hydrolysis time of 80 - 160 min, and boiling to inactivate the enzyme for 5 - 10 min after hydrolysis; The hydrolysis conditions of the acid protease are a temperature of 40 - 55 °C, adjusting the pH to a range of 2.5 - 3.5 with acetic acid, a hydrolysis time of 60 - 120 min, and inactivating the enzyme at 80 - 90 °C for 3 - 6 min after hydrolysis; The acid protease is 3.350 acid protease; The polypeptides from scallops B are prepared by first pretreating scallop slurry with aspergillus lipase to obtain a pretreatment solution, and then subjecting the pretreatment solution to two hydrolyses with 3.4310 acid protease and 537 acid protease in sequence, followed by centrifugation to remove residues, filtration, and ultrafiltration to obtain a polypeptide solution with a molecular weight cut-off below 1000 Da, and then spray-drying into powder; The hydrolysis conditions of the 3.4310 acid protease are adjusting the pH to 2.5 - 3.5 with acetic acid, a temperature of 40 - 52 °C, a hydrolysis time of 80 - 160 min, and raising the temperature to 80 - 90 °C to inactivate the enzyme for 3 - 5 min after hydrolysis; The hydrolysis conditions of the 537 acid protease are adjusting the pH to 2.5 - 3.5 with acetic acid, a temperature of 45 - 58 °C, a hydrolysis time of 60 - 140 min, and inactivating the enzyme at 80 - 90 °C for 3 - 6 min after hydrolysis.

2. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: The preparation method of the scallop slurry is to mix scallops and water at a mass ratio of 1:10 - 15, heat to 80 - 90 °C, and keep warm and soften for 1 - 3 hours; then pour it into a colloid mill and circulate for grinding for 2 - 3 minutes to form scallop slurry.

3. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the polypeptides from scallops A, calculated based on the protein content in the scallop slurry used, the addition amount of papain is 4000 - 8000 u / g, and the addition amount of acid protease is 3000 - 7000 u / g.

4. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the polypeptides from scallops A, after the scallop hydrolyzate after two enzymatic hydrolyses is centrifuged at 4000 - 6000 rpm to remove residues, the obtained supernatant is microfiltered through a 0.22 - 0.45 μm membrane, the microfiltered solution is ultrafiltered through a 1000 Da ultrafiltration membrane, and the filtrate is nanofiltrated through a 200 Da nanofiltration membrane and concentrated to a polypeptide content of 5% - 7% to obtain a nanofiltration concentrated polypeptide solution with a molecular weight of 200 - 1000 Da.

5. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the polypeptides from scallops A, the polypeptide solution after nanofiltration concentration is also vacuum concentrated and acetic acid is removed before spray drying to make the pH value reach 5.0 - 6.5, and when the polypeptides from scallops A prepared after spray drying are diluted to a polypeptide content of 0.4% - 1.8%, the inhibition rate against α-amylase reaches 38% - 65%.

6. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the scallop polypeptide B, calculated based on the protein content in the scallop slurry used, the addition amount of 3.4310 acidic protease is 4000 - 8000 u / g, and the addition amount of 537 acidic protease is 3000 - 7000 u / g.

7. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the scallop polypeptide B, the scallop hydrolyzate after three enzymatic hydrolyses is first centrifuged at 4000 - 6000 rpm to remove the precipitate residue; the obtained supernatant is filtered with diatomaceous earth, and the filtrate is ultrafiltered with a 1000 Da ultrafiltration membrane to obtain a polypeptide solution with a molecular weight less than 1000 Da.

8. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the scallop polypeptide B, the polypeptide solution ultrafiltered through the ultrafiltration membrane is also vacuum concentrated and acetic acid is removed before spray drying to make the pH value reach 5.0 - 6.5, and when the prepared scallop polypeptide B is diluted to a polypeptide content of 0.5% - 3.0% after spray drying, the inhibition rate against pancreatic lipase is 40% - 75%.

9. The preparation method of the fan shell polypeptide with weight loss function according to claim 1, characterized in that: In the preparation of the scallop polypeptide B, the addition amount of the aspergillus lipase is 0.3% - 0.7% of the scallop quality used. The pretreatment conditions of the aspergillus pancreatic lipase are to adjust the pH to 6.5 - 8.0 with a calcium hydroxide emulsion and treat at a temperature of 40 - 50 °C for 60 - 120 min.

10. A fan shell polypeptide with a weight loss function, characterized in that: It is prepared by using the preparation method of the scallop polypeptide with a weight loss function according to any one of claims 1 to 9.

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