A method for extracting bird's nest acid by ultrasonic combined enzymatic hydrolysis

Abstract

The present invention relates to a method for extracting bird's nest acid by ultrasonic combined enzymatic hydrolysis, comprising the following steps: soaking bird's nest pieces and bird's nest corners; crushing wet materials; ultrasonic crushing; enzymatic hydrolysis; enzyme inactivation; centrifugation and filtration. The method of the present invention recycles bird's nest pieces and bird's nest corners formed during the transportation and preparation of bird's nests, extracts sialic acid, protein and other nutritional components from bird's nest pieces and bird's nest corners through the steps of water swelling, wet crushing, ultrasonic crushing, enzyme extraction, filtration and the like, and obtains a high-sialic acid extract.

Description

Technical Field

[0001] The invention relates to the technical field of bird's nest preparation, and in particular to a method for extracting bird's nest acid by combining ultrasound and enzymatic hydrolysis. Background Art

[0002] Bird's nest refers to the saliva secreted by some swifts of the Apodidae family and several swiftlets of the genus Swiftlet, mixed with other substances to build a nest. It is also called bird's nest, bird's root, and bird's vegetable. Bird's nest mainly contains protein, sugar, lipid, some inorganic elements, vitamins, hormones, etc. A study analyzed the amino acids contained in bird's nest and found that it contains 20 common amino acids such as aspartic acid and glutamic acid, does not contain hydroxyproline and sarcosine, but contains 8 essential amino acids. The content of sugars in bird's nest is second only to protein, mainly sialic acid (also known as bird's nest acid), mannose, glucosamine, galactosamine, etc. Lipids account for about 0.14-0.28% of bird's nest, and fatty acid components include stearic acid, palmitic acid, linolenic acid, linoleic acid, etc. The content of inorganic elements in bird's nest is relatively high, and it contains trace elements necessary for humans. Studies have shown that the elements with higher content in bird's nest are four major elements Ca>Mg>Na>K, followed by five essential trace elements Fe>Zn>Mn>Cr>Cu.

[0003] During the transportation and processing of bird's nest, about 15% of bird's nest will be broken and bird's nest corners. The nutritional value of these broken and bird's nest corners is as rich as that of bird's nest strips, but their use is limited by factors such as poor appearance or difficulty in soaking. They are often discarded or resold at low prices, which causes a great waste of resources. Therefore, exploring the reasonable disposal methods of broken and bird's nest corners is particularly important for improving the utilization rate of raw materials and even increasing the added value of enterprises. Summary of the invention

[0004] In view of this, the present invention provides a method for extracting bird's nest acid by combining ultrasound and enzymatic hydrolysis.

[0005] This method can broaden the scope of use of bird's nest pieces and bird's nest corners, and increase the added value of bird's nest raw materials. The extract can be sold separately as an oral liquid, or it can be added to other foods as a stock solution.

[0006] The present invention provides a method for extracting bird's nest acid by ultrasonic combined with enzymatic hydrolysis, comprising the following steps:

[0007] 1) Soak the chopped bird's nest: add 3-8 times the dry weight of the chopped bird's nest in pure water and soak;

[0008] 2) Wet material crushing: wet crushing the soaked bird's nest horns in step 1) into millimeter-sized particles;

[0009] 3) Ultrasonic crushing: ultrasonic crushing the ground bird's nest corners in step 2);

[0010] 4) Enzymatic hydrolysis: adding alkaline protease to the broken bird's nest after ultrasonication in step 3) for enzymatic hydrolysis;

[0011] 5) Inactivation of enzyme: heating the enzymatic sample to inactivate the protease;

[0012] 6) Centrifugation and filtration: The enzymatic hydrolyzate from step 5) is placed in a centrifugal device for treatment, filtered to obtain a supernatant, and then passed through two vibrating sieves in sequence to obtain an extract;

[0013] Preferably, in step 1), the soaking water temperature is 20-30° C., the soaking time is 3-8 hours, the pH value is 6.0-9.0, and the conductivity is ≤10 μs / cm.

[0014] Preferably, in step 2), the wet grinding speed is 300-800 r / min, the time is 10-25 min, and the crushed particle size is 0.25-0.50 mm to obtain a better ultrasonic crushing effect.

[0015] Preferably, the ultrasonic power used in the ultrasonic crushing in step 3) is 50-200W, the ultrasonic frequency is 15-40KHz, and the ultrasonic time is 15-35min. Ultrasonic crushing can improve the solubility of the bird's nest and enhance the cleavage of structural bonds.

[0016] Preferably, the protease in step 4) is a serine endoprotease, and the added amount is 4‰ to 10‰ of the dry matter weight of the broken bird's nest, the enzymatic hydrolysis conditions are 45-60°C, the enzymatic hydrolysis time is 3.5-6.5h, and the solid-liquid ratio is 2:100-5:100 (g / ml).

[0017] Preferably, the enzyme inactivation conditions in step 5) are 85-95° C. and the time is 15-45 min.

[0018] Preferably, the centrifugal speed in step 6) is 3500-6500 r / min, and the time is 5-10 min; the supernatant is passed through 40-mesh and 200-mesh sieves in sequence.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The use scope of bird's nest crushes and bird's nest corners has been maximized, and the cavitation effect and mechanical effect of ultrasound have been used to increase the rate and frequency of molecular movement, improve the solubility of bird's nest crushes, and promote the dissolution of the components to be extracted during the hydrolysis process; the collision, friction, and impact of the grinding media in the ball mill with the material and the tank wall are used to crush the material into uniform and fine particles, increasing the contact area between the material and water and improving the hydrolysis efficiency. DETAILED DESCRIPTION

[0021] To further illustrate the present invention, specific description is given in conjunction with the following examples. Unless otherwise specified, the reagents used in the implementation of the present invention are all commercially available products, and the databases used in the implementation of the present invention are all public online databases. The following examples are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0022] Example 1

[0023] 1) Soaking the bird's nest pieces: Soak the bird's nest pieces with 5 times the amount of pure water, the water temperature is 25℃, the time is 6 hours, the pH is 8.0, the conductivity is ≤10μs / cm, and the stirring frequency is 1 time / 30min.

[0024] 2) Wet material crushing: Place the soaked bird's nest pieces prepared in step 1) in a crushing device with a rotation speed of 600r / min and wet crush for 10 minutes until the particle size is about 0.25mm.

[0025] 3) Ultrasonic crushing: Place the ground bird's nest in step 2) in an ultrasonic device with an ultrasonic intensity of 150 W, an ultrasonic frequency of 20 KHz, and an ultrasonic time of 30 min.

[0026] 4) Enzymatic hydrolysis: add alkaline protease (serine endoprotease) to the bird's nest pieces treated in step 3) for enzymatic hydrolysis; the amount of enzyme added is 8‰ of the dry bird's nest pieces, the enzymatic hydrolysis conditions are 55°C, the enzymatic hydrolysis time is 5.5h, and the material-liquid ratio is 3:100g / ml.

[0027] 5) Inactivation of enzyme: heating the enzymatic hydrolysis sample in step 4) to inactivate the protease; the enzyme inactivation conditions are 85° C. and the time is 30 min.

[0028] 6) Centrifugation and filtration: The enzymatic hydrolysate from step 5) is centrifuged at 4500 r / min for 8 min, and then passed through 40-mesh and 200-mesh vibrating sieves in sequence to obtain a highly concentrated clear solution, which is the extract.

[0029] Example 2: Comparative Example

[0030] 1) Soaking the bird's nest pieces: Soak the bird's nest pieces with 5 times the amount of pure water, the water temperature is 25℃, the time is 6 hours, the pH is 8.0, the conductivity is ≤10μs / cm, and the stirring frequency is 1 time / 30min.

[0031] 2) Wet material crushing: Place the soaked bird's nest pieces prepared in step 1) in a crushing device with a rotation speed of 600r / min and wet crush for 10 minutes until the particle size is about 0.25mm.

[0032] 3) Ultrasonic crushing: Place the ground bird's nest in step 2) in an ultrasonic device with an ultrasonic intensity of 200 W, an ultrasonic frequency of 15 KHz, and an ultrasonic time of 30 min.

[0033] 4) Hydrolysis: Add purified water to the bird's nest crushed treated in step 3), the amount of water added is 8‰ of the dry bird's nest crushed, the hydrolysis conditions are 55°C, the time is 5.5h, and the solid-liquid ratio is 3:100g / ml.

[0034] 5) Centrifugation and filtration: The enzymatic hydrolysate from step 5) is centrifuged at 4500 r / min for 8 min, and then passed through 40-mesh and 200-mesh vibrating sieves in sequence to obtain a highly concentrated clear solution, which is the extract.

[0035] Example 3: Comparative Example

[0036] 1) Soaking the bird's nest pieces: Soak the bird's nest pieces with 5 times the amount of pure water, the water temperature is 25℃, the time is 6 hours, the pH is 8.0, the conductivity is ≤10μs / cm, and the stirring frequency is 1 time / 30min.

[0037] 2) Wet material crushing: Place the soaked bird's nest pieces prepared in step 1) in a crushing device with a rotation speed of 600r / min and wet crush for 10 minutes until the particle size is about 0.25mm.

[0038] 3) Enzymatic hydrolysis: add alkaline protease (serine endoprotease) to the bird's nest pieces treated in step 2) for enzymatic hydrolysis; the amount of enzyme added is 8‰ of the dry bird's nest pieces, the enzymatic hydrolysis conditions are 55°C, the enzymatic hydrolysis time is 5.5h, and the solid-liquid ratio is 3:100g / ml.

[0039] 4) Inactivation of enzyme: heating the enzymatic hydrolysis sample in step 3) to inactivate the protease; the enzyme inactivation conditions are 85° C. and the time is 30 min.

[0040] 5) Centrifugation and filtration: The enzymatic hydrolysate from step 4) is centrifuged at 4500 r / min for 8 min, and then passed through 40-mesh and 200-mesh vibrating sieves in sequence to obtain a highly concentrated clear solution, which is the extract.

[0041] Example 4

[0042] 1) Soaking the bird's nest pieces: Soak the bird's nest pieces with 5 times the amount of pure water, the water temperature is 25℃, the time is 6 hours, the pH is 7.0, the conductivity is 10-20μs / cm, and the stirring frequency is 1 time / 30min.

[0043] 2) Wet material crushing: Place the soaked bird's nest pieces prepared in step 1) in a crushing device with a rotation speed of 300r / min and wet crush for 10 minutes until the particle size is about 0.50mm.

[0044] 3) Ultrasonic crushing: Place the ground bird's nest in step 2) in an ultrasonic device with an ultrasonic intensity of 100 W, an ultrasonic frequency of 40 KHz, and an ultrasonic time of 20 min.

[0045] 4) Enzymatic hydrolysis: add alkaline protease (serine endoprotease) to the bird's nest pieces soaked in step 3) for enzymatic hydrolysis; the amount of enzyme added is 5‰ of the dry bird's nest pieces, the enzymatic hydrolysis conditions are 60°C, the enzymatic hydrolysis time is 4h, and the material-liquid ratio is 3:100g / ml.

[0046] 5) Inactivation of enzyme: heating the enzymatic hydrolysis sample in step 4) to inactivate the protease; the enzyme inactivation conditions are 85° C. and the time is 30 min.

[0047] 6) Centrifugation and filtration: The enzymatic hydrolysate from step 5) is centrifuged at 4500 r / min for 8 min, and then passed through 40-mesh and 200-mesh vibrating sieves in sequence to obtain a highly concentrated clear solution, which is the extract.

[0048] According to the existing measurement standards and methods, the sialic acid extracts prepared in Examples 1 to 4 were measured. The protein content of the extracts was tested according to GB5009.5-2016, the sialic acid content of the extracts was tested according to GB / T30636-2014, and the total number of colonies of the extracts was tested according to GB4789.2-2016. The results are shown in Table 1.

[0049] Table 1 Nutrient content of extract

[0050]

[0051] At the same time, in order to ensure that the extract can be used directly, the extract was tested for nitrite, heavy metals, microorganisms and other indicators in accordance with the enterprise standard QB / BZXXD 0001-2021. The results are shown in Table 2.

[0052] Table 2 Safety index content data table of extract

[0053]

[0054]

[0055] It can be seen from Table 1 that the content of bird's nest acid and protein extracted by ultrasound-assisted enzymatic hydrolysis (Example 1) are 61.3% and 75.5% higher than those by water extraction (Example 2), respectively; it can be seen from Example 3 that the content of bird's nest acid and protein using ultrasound-assisted extraction (Example 1) are 21.1% and 19.0% higher than those without ultrasound (Example 3), respectively; in addition, the particle size of the bird's nest shreds, ultrasonic parameters, enzymatic hydrolysis conditions, etc. will also affect the extraction efficiency. The bird's nest acid and protein contents of Example 1 (optimal) are increased by 24.6% and 25.0% respectively compared with those of Example 4.

[0056] At the same time, several indicators in Table 2 show that the safety of the extract meets the standards and can be used directly as the original extraction solution.

[0057] In summary, the present invention grinds and crushes bird's nest pieces and bird's nest corners to increase the contact area between the substrate and the enzyme, improve the solubility of bird's nest pieces, and promote the efficiency of enzymolysis. It also utilizes the cavitation effect and mechanical effect of ultrasound to increase the rate and frequency of molecular movement, improve the solubility of bird's nest pieces, promote the dissolution of the components to be extracted during the hydrolysis process, and improve the efficiency of enzymolysis. This method can broaden the scope of use of bird's nest pieces and bird's nest corners, increase the added value of bird's nest raw materials, and the extract can be sold separately as an oral liquid or added to other foods as a sialic acid stock solution.

[0058] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and conversions can be made without exceeding the spirit and scope of the present invention defined by the appended claims. Moreover, the scope of the present application is not limited to the specific embodiments of the process, equipment, means, method and steps described in the specification. It will be easily understood by those of ordinary skill in the art from the disclosure of the present invention that the process, equipment, means, method or step that performs the function substantially the same as the corresponding embodiment described herein or obtains the result substantially the same as the corresponding embodiment described herein, existing and future to be developed can be used according to the present invention. Therefore, the appended claims are intended to include such processes, equipment, means, methods or steps within their scope.

Claims

1. A method for extracting bird's nest acid by ultrasonic combined with enzymatic hydrolysis, characterized in that: The following steps are involved: 1) Soak the chopped bird's nest: add 3-8 times the dry weight of the chopped bird's nest in pure water and soak; 2) Wet material crushing: wet crushing the soaked bird's nest horns in step 1) into millimeter-sized particles; 3) Ultrasonic crushing: ultrasonic crushing the ground bird's nest corners in step 2); 4) Enzymatic hydrolysis: adding alkaline protease to the broken bird's nest after ultrasonication in step 3) for enzymatic hydrolysis; 5) Inactivation of enzyme: heating the enzymatic sample to inactivate the protease; 6) Centrifugation and filtration: The enzymatic hydrolyzate from step 5) is placed in a centrifugal device for treatment, filtered to obtain a supernatant, and then passed through two vibrating sieves in sequence to obtain an extract; In the step 1), the soaking water temperature is 20-30° C., the soaking time is 3-8 hours, the pH value is 6.0-9.0, and the conductivity is ≤10 μs / cm; In the step 2), the speed of wet grinding is 300-800 r / min, the time is 10-25 min, and the size of the crushed particles is 0.25-0.50 mm; The ultrasonic power used in the ultrasonic crushing in step 3) is 50-200W, the ultrasonic frequency is 15-40KHz, and the ultrasonic time is 15-35min; In the step 4), the alkaline protease is a serine endoprotease, and the amount added is 4‰ to 10‰ of the dry matter weight of the broken bird's nest, the enzymatic hydrolysis conditions are 45-60°C, the enzymatic hydrolysis time is 3.5-6.5h, and the solid-liquid ratio is 2:100g / ml-5:100g / ml.

2. The method according to claim 1, characterized in that The conditions for heating and inactivating the enzyme in step 5) are a temperature of 85-95° C. and a time of 15-45 min.

3. The method according to claim 1, characterized in that In the step 6), the centrifugal speed is 3500-6500 r / min and the time is 5-10 min.

4. The method according to claim 1, characterized in that In step 6), the supernatant is passed through 40-mesh and 200-mesh vibrating sieves in sequence.

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