A kind of bird's nest polypeptide and preparation method thereof

Abstract

The present invention relates to the technical field of deep processing of bird's nests, and in particular to a bird's nest polypeptide and a preparation method thereof. The preparation method comprises the steps of soaking the bird's nest in water, pre-hydrolyzing the bird's nest with an enzyme and an acid, and then hydrolyzing the pre-hydrolyzed bird's nest liquid with two acidic proteases in sequence, and then performing enzyme inactivation, centrifugation to remove residues, ultrafiltration and nanofiltration to obtain the bird's nest polypeptide. The method is characterized in that the bird's nest protein in the bird's nest has a high hydrolysis rate, and the feather impurities therein are not hydrolyzed. The bird's nest polypeptide obtained by the method is a bird's nest protein hydrolyzate and does not contain feather hydrolyzate. Therefore, the method provided by the present invention can simplify the feather picking process, use low-value bird's nests with feather impurities to make high-value bird's nest polypeptides, and improve the economic value of the bird's nests with feather impurities.

Description

Technical Field

[0001] The present invention relates to the technical field of bird's nest deep processing, and in particular to a bird's nest polypeptide and a preparation method thereof. Background Art

[0002] Bird's nest is a nest made by swiftlets and other swiftlets of the same genus using saliva and down feathers to condense. The protein in bird's nest is mainly glycoprotein, with a crude protein content of 50% to 60% and a bird's nest acid content of about 10%. The collected natural bird's nest contains a lot of feathers, and in the process of processing bird's nest food, it needs to go through a feather picking process. The leftovers after the bird's nest is picked still contain a certain amount of broken bird's nests. It is difficult to completely remove the feathers in it using conventional processes, so the broken bird's nests containing a lot of bird feathers and feather impurities are often dumped as waste. There are also patents or literature reports on the use of protease to hydrolyze bird's nests with feather impurities. For example, the patent document with application number CN202010892221 discloses a method for preparing bird's nest peptides, which uses collagenase, trypsin and chymotrypsin compound enzymes to hydrolyze bird's nests, but there is no record of using bird's nests without bird feathers for hydrolysis for comparative tests.

[0003] At present, the proteases reported for enzymatic hydrolysis of bird's nest polypeptides include collagenase, pepsin, trypsin, chymotrypsin, alkaline protease, neutral protease, papain, bromelain, flavor enzyme and fig protease, but there are no reports on the hydrolysis of bird's nest polypeptides by acidic protease, which may be related to the low yield of polypeptides directly hydrolyzed by acidic protease. There are also reports on the use of protease to hydrolyze bird's nests and then acid hydrolyze them to obtain small molecule bird's nest peptides. For example, the patent document with application number CN202010414309 discloses a method for simultaneously extracting bird's nest polypeptides and bird's nest polysaccharides from bird's nests, first hydrolyzing glycosidic bonds with protease, and obtaining supernatant and precipitate by salting out and centrifugation, and the supernatant is treated to obtain bird's nest polysaccharides; after the precipitate is redissolved in water, it is enzymatically hydrolyzed for 3 hours at room temperature, and then the pH of the solution is adjusted to 1-3 by citric acid, and acid hydrolyzed for 3-10 hours at 80-110°C, and the hydrolyzate is centrifuged and filtered through a membrane to obtain low molecular weight bird's nest polypeptides. For example, in the patent document "Bird's Nest Oligopeptide Composition, Preparation Method and Use thereof" with application number CN201010571507, it is proposed to first hydrolyze with protease, then adjust the pH to 1.5-2 with 10% hydrochloric acid, and hydrolyze at 80-90°C for 2-4 hours to obtain a nano oligopeptide composition containing small molecules.

[0004] Since the protein in the bird's nest is a glycoprotein, in order to obtain the maximum amount of polypeptides to facilitate the hydrolysis of proteases, it is necessary to decompose the glycopeptide bonds bound to the protein and the glycosidic bonds of the polysaccharides. Existing reports on the hydrolysis of bird's nest glycosidases include a method for hydrolyzing bird's nest peptide products disclosed in the patent document with application number CN202010365628, which mentions the use of pectinase or galactosidase to assist hydrolysis, and a method for preparing bird's nest polypeptides disclosed in the patent document with application number CN2016107929662, which assists hydrolysis by adding at least one of lysozyme, cellulase, and chitosanase. Summary of the invention

[0005] In order to prepare a bird's nest polypeptide with a higher polypeptide yield in the presence of feather impurities, the present invention provides the following method for preparing a bird's nest polypeptide, comprising the following preparation steps:

[0006] S100, pre-hydrolysis of bird's nest with feather impurities: soaking the bird's nest with feather impurities in water, then adjusting the pH of the soaking solution to 5.0-6.5 with organic acid, adding deaminase for enzyme treatment, and then adjusting the pH of the solution to 2.2-2.8 with organic acid and pre-hydrolyzing, so that the viscosity of the pre-hydrolyzed solution is reduced by more than 90%;

[0007] S200, first protease hydrolysis: adding Bacillus licheniformis acid protease to the pre-hydrolyzed solution obtained in step S100 for first hydrolysis to obtain a first protease hydrolyzed solution;

[0008] S300, second protease hydrolysis: adding 3.350 or 3.4310 acid protease to the first protease hydrolyzate obtained in step S200 for a second hydrolysis to obtain a second protease hydrolyzate;

[0009] S400, polypeptide classification and drying: the second protease hydrolyzate after enzyme inactivation is centrifuged at 5000-8000rpm to remove residues, the supernatant is first ultrafiltered with a 10000Da ultrafiltration membrane, the filtrate obtained is then nanofiltered with a 200Da nanofiltration membrane and concentrated, and then spray-dried to obtain the bird's nest polypeptide.

[0010] In one embodiment, the first hydrolysis is carried out at pH 2.2-3.5 and 35-45° C. for 90-150 min, then the temperature is raised to 55° C., kept for 4-7 min, and then cooled to obtain the first protease hydrolyzate. After the hydrolysis is completed, the temperature is only raised to 55° C., and the enzyme is not completely inactivated, in order to retain a small amount of Bacillus licheniformis acid protease activity in the subsequent hydrolysis step.

[0011] In one embodiment, the second hydrolysis is carried out at pH 2.5-3.5 and 45-55° C. for 60-120 min to obtain a second protease hydrolyzate.

[0012] In one embodiment, the second protease hydrolyzate is inactivated at 80-90° C. for 10-20 min.

[0013] In one embodiment, the deaminase enzyme treatment condition is to keep the enzyme treatment at 50-60° C. for 2-4 hours.

[0014] In one embodiment, based on the mass of the bird's nest with feather impurities, the added amount of the deaminase is 300-600u / g; the added amount of the Bacillus licheniformis acid protease is 2000-4000u / g; the added amount of the 3.350 acid protease or 3.4310 acid protease is 2000-4000u / g.

[0015] In one embodiment, the soaking conditions of the bird's nest with feather impurities are a solid-liquid ratio of 1:20-40, and after soaking at room temperature for 8-12 hours, it is heated to 97-100° C. and then cooled to 50-60° C. within 70-100 min.

[0016] In one embodiment, the organic acid is acetic acid or lactic acid.

[0017] In one embodiment, the pre-hydrolysis condition is 85-95° C. for 2-4 hours.

[0018] In one embodiment, the polypeptide content in the bird's nest polypeptide is 68% to 72%, and the content ratio of the polypeptide to bird's nest acid is 6.0 to 6.4:1.

[0019] The present invention also provides a bird's nest polypeptide, which is prepared by any of the above-mentioned methods for preparing the bird's nest polypeptide.

[0020] Based on the above, compared with the prior art, the method for preparing a bird's nest polypeptide provided by the present invention has the following beneficial effects:

[0021] (1) After the bird's nest with feather impurities is heated to 97-100°C at room temperature, it is slowly cooled to 50-60°C within 70-100 min, which is conducive to the full swelling of the bird's nest and the subsequent hydrolysis of polypeptides; at the same time, a suitable high temperature environment is controlled to avoid excessive softening of feather keratin caused by long-term high temperature, which leads to its hydrolysis by the subsequent action of acid and enzyme.

[0022] (2) Using deaminase and pH 2.2-2.8 organic acid solution to pre-degrade bird's nest glycoproteins in bird's nests can not only destroy the glycopeptide bonds and glycosidic bonds in polyglycoproteins, but also will not cause the hydrolysis of keratin. In the early experiments, it was accidentally discovered that after adding a small amount of deaminase to the swollen bird's nest slurry for enzyme treatment and then soaking it in a hot organic acid aqueous solution for a certain period of time, the viscosity of the bird's nest slurry can be reduced by more than 90%; the viscosity reduction rate is much higher than that of the bird's nest slurry not treated with deaminase, indicating that the deaminase treatment may cause the amino sugars or glycoproteins in the bird's nest glycoproteins to produce deamination reactions, thereby destroying the structural stability of the glycoproteins. The glycopeptide bonds and glycosidic bonds of the polyglycoproteins are more easily degraded in the subsequent high-temperature acidic aqueous solution, which significantly reduces the viscosity of the slurry.

[0023] (3) According to relevant literature reports, keratin can be hydrolyzed by pepsin, trypsin, papain, chymotrypsin, Aspergillus oryzae and Bacillus subtilis under certain conditions, and can also be hydrolyzed by 1% to 2% hydrochloric acid solution. If the appropriate enzyme, hydrolysis pH and temperature conditions are not screened, it is possible to cause the hydrolysis of bird's nest feathers, so that the enzymatic hydrolysis product becomes a hydrolysis mixture of bird's nest protein and feather keratin. The present invention has determined through a large number of experiments that the bird's nest is first treated with deaminase, and then pre-hydrolyzed with a milder organic acid solution at pH 2.2 to 2.8 and 85 to 95°C (no free amino acids are produced after detection); then the bird's nest pre-hydrolyzate with feather impurities is enzymatically hydrolyzed with two acidic proteases in sequence, and the obtained polypeptide content / bird's nest acid content ratio is basically the same as the content ratio of the two without bird feathers, and the feathers in the slurry remain intact, indicating that this method does not hydrolyze feather keratin. Therefore, the method provided by the present invention can simplify the feather picking process, use low-value bird's nests with feather impurities to make high-value bird's nest polypeptides, and improve the economic value of bird's nests with feather impurities.

[0024] (4) The present invention adopts a method of pre-hydrolyzing the bird's nest with feather impurities and then hydrolyzing it with two acidic proteases in sequence, so that the yield of bird's nest polypeptides can reach 90% to 93% of the bird's nest protein, which finds a new method for the deep processing of bird's nest with feather impurities.

[0025] Other features and beneficial effects of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other beneficial effects of the present invention can be achieved and obtained by the structures and / or components indicated in the description and claims. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0027] 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 generally understood by ordinary technicians in the field to which the present invention belongs, and cannot be understood as limiting the present invention; it should be further understood that the terms used in the present invention should be understood to have the same meanings as these terms in the context of this specification and in the relevant fields, and should not be understood in an idealized or overly formal sense, unless explicitly defined in the present invention.

[0028] The present invention provides the following embodiments

[0029] Example 1

[0030] S110. Pre-hydrolysis of bird's nest with feather impurities: add 3000 g of water to 100 g of bird's nest with feather impurities, soak at 15°C for 12 hours, heat to 100°C, put into hot water, and cool to about 60°C within 90 min; adjust the pH of the soaking liquid to 5.5 with lactic acid, add 40,000 u of deaminase according to the mass of the bird's nest with feather impurities, and keep warm at 60°C for 120 min; then adjust the pH of the bird's nest with feather impurities to 2.2 with lactic acid for acid hydrolysis, and preliminarily detect the viscosity of the liquid before acid hydrolysis to be 685 mPa·s, keep warm at 85°C for 4 hours, and detect the viscosity of the pre-hydrolyzed liquid after acid hydrolysis to be 55 mPa·s, and the pre-hydrolysis is completed.

[0031] S120, first protease hydrolysis: after the pre-hydrolyzate obtained in step S110 is cooled, 400,000 u of Bacillus licheniformis acid protease is added, and hydrolyzed for 150 min at pH 3.0 and 36° C. After enzymolysis, the temperature is raised to 55° C. and kept for 6 min, and then cooled to obtain the first protease hydrolyzate.

[0032] S130, second protease hydrolysis: add 200,000 u of 3.350 acidic protease to the first protease hydrolysate obtained in step S120, and perform a second hydrolysis at pH 2.5 and 53° C. for 60 min; after the hydrolysis, the second protease hydrolysate obtained is heated to 90° C. for 10 min to inactivate the enzyme.

[0033] S140, polypeptide classification and drying: the second protease hydrolyzate after enzyme inactivation is centrifuged at 5000rpm for 15min to remove residues, the supernatant is ultrafiltered with a 10000Da ultrafiltration membrane, the filtrate is nanofiltered with a 200Da nanofiltration membrane to remove small molecules such as inorganic salts, ammonia and monosaccharides, and then concentrated and spray-dried into polypeptide powder.

[0034] In order to better illustrate the pre-hydrolysis of the present invention, the bird's nest is first treated with a deaminase, which is beneficial to the destruction of the glycopeptide bond or glycosidic bond by the subsequent acid hydrolysis of the polysaccharide protein, thereby greatly reducing the viscosity of the slurry. The present invention also provides the following comparative example 1:

[0035] Comparative Example 1

[0036] The only difference from Example 1 is that deaminase is not added in the pre-hydrolysis step, and the other operations are consistent with Example 1. The viscosity of the slurry before acid hydrolysis is detected to be 690 mPa·s, and the viscosity of the pre-hydrolyzed liquid obtained after acid hydrolysis is 355 mPa·s.

[0037] The polypeptide content and bird's nest acid content in the polypeptide powders prepared in Example 1 and Comparative Example 1 were detected and the polypeptide yield was calculated. The polypeptide content was detected according to the method of "GB / T22492-2008 Soybean Peptide Powder", and the bird's nest acid content was detected according to the method of "GB / T30636-2014 Determination of Sialic Acid in Bird's Nest and Its Products". The results are shown in Table 1:

[0038] Table 1

[0039]

[0040] It can be seen from the test results in Table 1 that the bird's nest with feather impurities in Comparative Example 1 was not treated with deaminase before pre-hydrolysis, and the polypeptide content and bird's nest acid content in the obtained polypeptide powder were much lower than those in Example 1, and the polypeptide yield relative to the raw material was also very low; from the change in viscosity before and after acid hydrolysis, the viscosity of Comparative Example 1 after acid hydrolysis was only reduced by 39.83% compared with that before hydrolysis, while that of Example 1 was reduced by 91.28%, indicating that after the bird's nest slurry of Example 1 was treated with deaminase and then hydrolyzed with a weak organic acid, the glycopeptide bonds and glycosidic bonds of the polysaccharide protein were cleaved more, so the viscosity decreased significantly; while the bird's nest slurry of Comparative Example 1 was not treated with deaminase, and the viscosity decreased less during organic acid hydrolysis, indicating that since the glycoprotein had no deamination reaction, there might only be acid hydrolysis of the glycosidic bond, so the viscosity decreased less.

[0041] Example 2

[0042] S210. Pre-hydrolysis of bird's nest with feather impurities: add 4000 grams of water to 100 grams of bird's nest with feather impurities, soak at 20°C for 8 hours, heat to 98°C, put into hot water, and cool to 50°C within 100 minutes; take samples to detect free amino nitrogen in the soaking slurry, then adjust the pH to 6.0 with acetic acid, add 60,000 U of deaminase, and keep warm at 50°C for 240 minutes; adjust the pH of the bird's nest with feather impurities to 2.5 with acetic acid for acid hydrolysis, detect the viscosity of the slurry before acid hydrolysis, keep pre-hydrolyzed at 95°C for 2 hours, detect the viscosity of the pre-hydrolyzed liquid after acid hydrolysis, and the pre-hydrolysis is completed.

[0043] S220, first protease hydrolysis: After the pre-hydrolyzate obtained in step S210 is cooled, 250,000 u of Bacillus licheniformis acid protease is added, and hydrolyzed for 90 min at pH 3.5 and 45° C. After enzymolysis, the temperature is raised to 55° C. and kept for 4 min, and then cooled to obtain the first protease hydrolyzate.

[0044] S230, second protease hydrolysis: add 400,000 u of 3.4310 acidic protease to the first protease hydrolysate obtained in step S220, and perform a second hydrolysis at pH 3.0 and 50°C for 100 min; after the hydrolysis is completed, take a sample to detect the amino nitrogen in the hydrolysate; heat the obtained second protease hydrolysate to 80°C for 20 min to inactivate the enzyme.

[0045] S240, polypeptide classification and drying: the second protease hydrolyzate after enzyme inactivation is centrifuged at 7000 rpm for 10 min to remove residues, the supernatant is ultrafiltered with a 10000Da ultrafiltration membrane, the filtrate is nanofiltered with a 200Da nanofiltration membrane and concentrated, and then spray-dried into polypeptide powder.

[0046] In order to better illustrate that the preparation method of the present invention does not hydrolyze bird feathers, the present invention provides Comparative Examples 2 and 3:

[0047] Comparative Example 2

[0048] 100 g of pure bird's nest without bird feathers (the crude protein content of the bird's nest is 52.1%) is used to replace the bird's nest with feather impurities in Example 2 for pre-hydrolysis, and the remaining operations are consistent with Example 2.

[0049] Comparative Example 3

[0050] Take 100 grams of the same pure bird's nest as in Comparative Example 2 (the crude protein content of the bird's nest is 52.1%) and add 20 grams of swiftlet feathers to replace the bird's nest with feather impurities in Example 2. The subsequent amount of linker enzyme added is based on 100 grams of pure bird's nest. Other operating conditions and processes are the same as in Example 2.

[0051] The amino nitrogen content detected during the preparation of Example 2, Comparative Example 2 and Comparative Example 3 is shown in Table 2; the detection and calculation results of the polypeptide content, bird's nest acid content and polypeptide yield in each polypeptide powder sample are shown in Table 2.

[0052] Table 2

[0053]

[0054] Note: Comparative Example 3 has too many feathers and the viscosity was not measured

[0055] It can be seen from the test results in Table 2 that, under the condition of the same amount of enzyme added, the polypeptide content and the content of saccharide in the polypeptide powder prepared in Example 2 are slightly higher than those in Comparative Example 2. The reason may be that the content of bird's nest protein in the raw material of Comparative Example 2 without feathers is higher than that in Example 2, and the amount of protease acting on the bird's nest protein is less than that in Example 2, and the enzymatic effect is weakened. Therefore, the content of polypeptide and saccharide in Comparative Example 2 is lower than that in Example 2. However, the ratio of polypeptide content to saccharide content in Comparative Example 2 and Example 2 is very similar. Since the hydrolysis of bird's nest protein is inevitably accompanied by a synchronous and proportional increase in the content of polypeptide and saccharide, if the ratio does not increase significantly, it can be explained that the feathers in the raw material of Example 2 have not been hydrolyzed into polypeptides.

[0056] As can be seen from Table 2, before and after acid hydrolysis, the viscosity of Example 2 and Comparative Example 2 decreased by 91.40% and 92.14%, respectively. This may be caused by the cleavage of glycosidic bonds and glycopeptide bonds in the bird's nest during acid hydrolysis after deaminase treatment.

[0057] By analyzing Comparative Example 3, it can be seen that the polypeptide content and bird's nest acid content of the polypeptide powder obtained by adding bird's nest feathers to pure bird's nest are slightly lower than those in Example 2, indicating that the keratin in Comparative Example 3 has not been hydrolyzed to produce additional polypeptides and bird's nest acid. The polypeptide content to bird's nest acid content ratio, polypeptide content and bird's nest acid content of Comparative Example 3 are very close to those of Comparative Example 2, which also shows that Example 3 with additional feathers added has the same hydrolysis effect as pure bird's nest without feathers.

[0058] The level of free amino acid nitrogen can also reflect the degree of hydrolysis of the raw material protein (including bird's nest protein and keratin), and the amino acid nitrogen content of the hydrolyzate of Comparative Example 3 with feathers added is basically the same as that of the pure bird's nest (Comparative Example 2), with a difference of only 0.03%. This shows that the addition of feathers does not affect the hydrolysis of normal bird's nests; conversely, it also shows that there is no sign of keratin being hydrolyzed into amino acids. In addition, after the comparative experiment, the inventor also used the process of Comparative Example 3 to observe the hydrolysis of all feathers, and no polypeptide was detected in the hydrolyzate, which also confirmed the result that the feathers were not hydrolyzed from another side.

[0059] The crude protein content in the bird's nests of Comparative Example 2 and Comparative Example 3 is 52.1%, while the polypeptide yield of Comparative Example 2 relative to the bird's nest is 47.76%, and the polypeptide yield of Comparative Example 3 relative to the pure bird's nest is 46.98%. It is calculated that the polypeptide conversion yield relative to the protein is 91.67% and 90.17% for Comparative Example 2 and Comparative Example 3, respectively. It can be seen that by using the bird's nest polypeptide hydrolysis method of the present invention, whether the raw material contains feather impurities or not, a polypeptide yield of more than 90% can be obtained.

[0060] Example 3

[0061] S310, pre-hydrolysis of bird's nest with feather impurities: add 2500 grams of water to 100 grams of bird's nest with feather impurities, soak at 18°C ​​for 10 hours, heat to 98°C, put into hot water, and cool to 55°C within 80 minutes; then adjust the pH to 5.0 with lactic acid, add 50,000 U of deaminase, and keep warm at 55°C for 180 minutes; adjust the pH of the bird's nest with feather impurities to 2.6 with acetic acid for acid hydrolysis, and test the viscosity of the pre-hydrolysis solution before acid hydrolysis, keep warm at 95°C for 3 hours, and test the viscosity of the pre-hydrolysis solution obtained after acid hydrolysis again, and the pre-hydrolysis is completed.

[0062] S320, first protease hydrolysis: after the pre-hydrolyzate obtained in step S310 is cooled, 300,000 u of Bacillus licheniformis acid protease is added, and hydrolyzed for 120 min at pH 2.3 and 40°C. After enzymolysis, the temperature is raised to 55°C and kept for 5 min, and then cooled to obtain the first protease hydrolyzate.

[0063] S330, second protease hydrolysis: add 300,000 u of 3.350 acidic protease to the first protease hydrolysate obtained in step S320, and perform a second hydrolysis at pH 3.2 and 45°C for 120 min; after the hydrolysis, the second protease hydrolysate obtained is heated to 85°C for 15 min to inactivate the enzyme.

[0064] S340, polypeptide classification and drying: the second protease hydrolysate after enzyme inactivation is centrifuged at 8000rpm for 5min to remove the residue, the viscosity of the enzymatic hydrolysis supernatant is tested, and then ultrafiltered with a 10000Da ultrafiltration membrane, the filtrate is nanofiltered and concentrated with a 200Da nanofiltration membrane, and then spray-dried into polypeptide powder.

[0065] In order to better illustrate that the preparation method provided by the present invention can effectively improve the yield of bird's nest polypeptide, the present invention also provides the following comparative examples 4 to 8:

[0066] Comparative Example 4

[0067] After the first protease hydrolysis was completed, the mixture was directly heated to 85° C. to inactivate the enzyme for 15 min without performing a second protease hydrolysis. Other operations and processes were the same as those in Example 3.

[0068] Comparative Example 5

[0069] The first protease hydrolysis step of using Bacillus licheniformis acid protease in Example 3 was removed, and other operations and processes were the same as those in Example 3.

[0070] Comparative Example 6

[0071] The order of the first protease hydrolysis and the second protease hydrolysis in Example 3 was reversed, that is, 3.350 acid protease hydrolysis was performed first without enzyme inactivation, and then Bacillus licheniformis acid protease hydrolysis was performed. After the two hydrolysis processes were completed, the enzyme was inactivated at 85°C for 15 min. The other operations and processes were the same as in Example 3.

[0072] Comparative Example 7

[0073] The only difference from Example 3 is that after the first protease hydrolysis, the enzyme is directly inactivated at 85°C for 15 min instead of keeping at 55°C for 5 min. Other operations and processes are the same as those in Example 3.

[0074] Comparative Example 8

[0075] The only difference from Example 3 is that the second protease hydrolysis is directly carried out after the first protease hydrolysis is completed, and there is no need to heat it to 55° C. and keep it for 5 min. Other operations and processes are the same as those in Example 3.

[0076] The polypeptide content and the bird's nest acid content in the polypeptide powders prepared in Example 3 and Comparative Examples 4 to 8 were detected and the polypeptide yield was calculated. The results are shown in Table 3:

[0077] Table 3

[0078]

[0079] From the test results in Table 3, it can be seen that the bird's nest slurry with feather impurities in Comparative Example 4 was hydrolyzed only with Bacillus licheniformis acid protease, without 3.350 acid protease hydrolysis, and the polypeptide content and polypeptide yield of the obtained sample were reduced by 40.9% compared with Example 3. While Comparative Example 5 was hydrolyzed only with 3.350 acid protease, without Bacillus licheniformis acid protease hydrolysis, the polypeptide content and polypeptide yield of the obtained sample were reduced by 62.34% compared with Example 3. The above two comparative examples show that the polypeptide yield is low when only one acid protease is used to hydrolyze the bird's nest.

[0080] The process of Comparative Example 6 is different from that of Example 3 in that the order of hydrolysis by the two acidic proteases is reversed, with 3.350 acidic protease hydrolyzed first and then with Bacillus licheniformis acidic protease hydrolyzed. The polypeptide yield is 33.28% lower than that of Example 3, indicating that the hydrolysis order is not good.

[0081] In Comparative Example 7, after the first protease hydrolysis, the enzyme was inactivated at 85°C instead of being kept at 55°C for 5 min and then cooled. As a result, the polypeptide yield was 5.28% lower than that of Example 3. This indicates that after the enzymatic hydrolysis of Bacillus licheniformis acid protease, the enzyme was not completely inactivated, and a small amount of enzyme activity was retained to participate in the hydrolysis of the second stage 3.350 acid protease, which was beneficial to improving the polypeptide yield.

[0082] In Comparative Example 8, after the first protease hydrolysis, the method of keeping warm at 55°C for 5 min (partial enzyme inactivation) was not adopted, and 3.350 g of acidic protease was directly added for the second protease hydrolysis. The polypeptide yield was 7.52% lower than that in Example 3, indicating that the acidic protease activity of Bacillus licheniformis remained too much, which may reduce the activity of the enzyme in the second protease hydrolysis.

[0083] By analyzing the viscosity changes of Example 3 and each comparative example before and after acid hydrolysis and after the second protease hydrolysis, it can be known that the more fully the bird's nest protein is hydrolyzed, the greater the decrease in viscosity, the smaller the final viscosity value of the enzymatic hydrolyzate, and the higher the polypeptide yield. Among them, in Example 3, after enzymatic hydrolysis, the viscosity of the supernatant decreased the most compared with that before acid hydrolysis, and the polypeptide yield was the highest, indicating that the bird's nest protein hydrolysis effect was the best.

[0084] In summary, the present invention uses deaminase and organic acid solution to pre-degrade bird's nest glycoprotein in bird's nest, which can not only destroy the glycopeptide bonds and glycosidic bonds in the polyglycoprotein, but also will not cause the hydrolysis of keratin. Then, two acidic proteases are used to successively enzymatically hydrolyze the bird's nest pre-hydrolyzate with feather impurities, and the obtained polypeptide content / bird's nest acid content ratio is basically the same as the content ratio of the two without bird feathers, and the feathers in the slurry remain intact, indicating that the two acidic protease treatments also do not hydrolyze feather keratin. Therefore, the method provided by the present invention can simplify the feather picking process. At the same time, the polypeptide yield of this method can reach 90% to 93% of the bird's nest protein, which greatly improves the extraction rate of bird's nest polypeptide.

[0085] 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 in only one or several aspects, without having to solve all the technical problems listed in the prior art or background technology at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be used as a limitation on the claim.

[0086] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing bird's nest polypeptide, characterized in that: The method comprises the following preparation steps: S100, pre-hydrolysis of bird's nest with feather impurities: soak the bird's nest with feather impurities in water, then adjust the pH of the soaking liquid to 5.0-6.5 with organic acid, add deaminase for enzyme treatment, and then adjust the pH of the liquid to 2.2-2.8 with organic acid before pre-hydrolysis, so that the viscosity of the pre-hydrolyzed liquid is reduced by more than 90%; The soaking conditions of the bird's nest with feather impurities are: a solid-liquid ratio of 1:20-40, soaking at room temperature for 8-12 hours, heating to 97-100° C., and then cooling to 50-60° C. within 70-100 minutes; The organic acid is acetic acid or lactic acid; The pre-hydrolysis condition is 85-95°C for 2-4 hours; The enzyme treatment condition of the deaminase is to keep the enzyme treatment at 50-60° C. for 2-4 hours; S200, first protease hydrolysis: adding Bacillus licheniformis acid protease to the pre-hydrolyzed solution obtained in step S100 for first hydrolysis to obtain a first protease hydrolyzate; S300, second protease hydrolysis: adding 3.350 or 3.4310 acidic protease to the first protease hydrolysate obtained in step S200 for a second hydrolysis to obtain a second protease hydrolysate; S400, polypeptide classification and drying: the second protease hydrolyzate after enzyme inactivation is centrifuged at 5000-8000rpm to remove residues, the supernatant is first ultrafiltered with a 10000Da ultrafiltration membrane, the filtrate obtained is then nanofiltered with a 200Da nanofiltration membrane and concentrated, and then spray-dried to obtain the bird's nest polypeptide.

2. The method for preparing the bird's nest polypeptide according to claim 1, characterized in that: The conditions for the first hydrolysis are: hydrolysis at pH 2.2-3.5, 35-45° C. for 90-150 min, then heating to 55° C., keeping the temperature for 4-7 min, and then cooling to obtain the first protease hydrolyzate.

3. The method for preparing the bird's nest polypeptide according to claim 1, characterized in that: The second hydrolysis is carried out at pH 2.5-3.5 and 45-55° C. for 60-120 min to obtain a second protease hydrolyzate.

4. The method for preparing bird's nest polypeptide according to claim 1, characterized in that: The enzyme inactivation condition of the second protease hydrolyzate is to inactivate the enzyme at 80-90° C. for 10-20 min.

5. The method for preparing bird's nest polypeptide according to claim 1, characterized in that: Calculated based on the mass of the bird's nest with feather impurities, the added amount of the deaminase is 300-600u / g; the added amount of the Bacillus licheniformis acid protease is 2000-4000u / g; and the added amount of the 3.350 acid protease or 3.4310 acid protease is 2000-4000u / g.

6. A bird's nest polypeptide, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 5.

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