CN111820412A

CN111820412A - Method for extracting cubilose, extract and product thereof

Info

Publication number: CN111820412A
Application number: CN202010797265.4A
Authority: CN
Inventors: 余的军; 石培松
Original assignee: Guangxi Free Trade Zone Yanhao Biotechnology Co ltd
Current assignee: Guangxi Free Trade Zone Yanhao Biotechnology Co ltd
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-10-27
Also published as: WO2022032803A1

Abstract

The application relates to a method for extracting cubilose, an extract and a product thereof. The method comprises the following steps: b, soaking clean cubilose powder to obtain cubilose pulp; c, adding water into the cubilose pulp in a weight ratio of 1-5 times of the cubilose pulp, and continuously stirring for 3-8 hours at 70-120 ℃ at 200-1000 rpm to obtain a first extracting solution; d, uniformly stirring the first extracting solution at the reduced temperature of 30-40 ℃, standing, removing a first lower layer liquid containing precipitates after impurities are precipitated, and taking out a supernatant as a second extracting solution; e, continuously stirring the residual liquid in the step D at 30-40 ℃, standing, and removing a second lower layer liquid containing precipitates after further impurities are precipitated to obtain a third extracting solution; and F, combining the second extracting solution and the third extracting solution to obtain the liquid cubilose extract. The method is mild, has no additional chemicals, and retains nutritional components in nidus Collocaliae; the low molecular weight peptide accounts for more than 45 wt% in the obtained extract, so that the absorption is facilitated, and meanwhile, the extraction rate of gao can reach more than 90 wt%.

Description

Method for extracting cubilose, extract and product thereof

Technical Field

The present invention relates to a method for extracting from bird's nest and various extracts obtained by the method.

Background

The traditional method of taking edible bird's nest as a tonic is long-standing in China. The record of cubilose is described in Jiajing, Chen 25035in the Ming dynasty of 1537, and written in Quannan journal. The efficacy of edible bird's nest was first recorded in Ben Jing Yuan (the origin of menstrual flow) of famous physicians in the Qing Dynasty.

More than half of the ingredients in the edible bird's nest are protein, and a certain proportion is water-soluble protein, wherein the protein comprises protein for promoting the growth of human tissues, 18 amino acids which are necessary and conditionally necessary for human bodies, and 8 essential amino acids. Cubilose acid in cubilose, also known as "N-acetylneuraminic acid (NeuAc)" for Sialic acid (Sialic acid), is an acetyl derivative of aminosaccharide neuraminic acid. Sialic acids have been found to have a number of beneficial effects on the nervous system, immune system, etc. In addition, the bird's nest also contains Epidermal Growth Factor (EGF) which has strong division promoting, proliferation, differentiation and regeneration effects on various histiocytes, has proliferation promoting and differentiation effects on hematopoietic stem cells in different development stages and Colony Stimulating Factor (CSF) resisting influenza virus, can be polymerized and extracted into glycoprotein for improving bone strength and dermis thickness, and can be polymerized and extracted into various glycoproteins for inhibiting or inducing cancer cell killing and stem cell vitality restoration. The contents of the proteins and sialic acid (and other components) become an important index for representing the nutritional value of the cubilose product.

The traditional way for people to eat cubilose is as follows: cleaning, soaking, washing, adding water, steaming and stewing. A significant portion of the water-soluble protein of the bird's nest has been lost during 2 to 3 washes, soaks, rinses and simmering. Because most of proteins in the cubilose are proteins with large molecular weight, the proteins are difficult to be changed into small-molecular proteins which can be easily and directly absorbed by a human body by the traditional eating mode.

At present, common edible bird's nest extract products or instant edible bird's nest products in the market are processed by adopting a medium-low temperature steaming and stewing processing mode, or adopting an enzymatic hydrolysis method or even a direct hydrolysis method. Among them, the enzymatic and hydrolysis methods may not only destroy the amino acids with poor stability and sialic acid in the form of glycoprotein, but also introduce additional chemicals, and these methods may lose part of nutrients, resulting in unsatisfactory extraction efficiency.

Therefore, there is still a need to improve the way in which the bird's nest is processed to better exploit the value of the bird's nest.

Disclosure of Invention

In view of the above, the main object of the present invention is to provide a method for extracting bird's nest and a bird's nest extract obtained by the method, wherein the method can retain nutrients in the bird's nest to a greater extent and decompose high molecular weight proteins into low molecular weight proteins having appropriate and more uniform molecular weights, thereby providing a more valuable bird's nest extract.

To this end, the present invention provides, in one aspect, a method for extracting bird's nest, the method comprising the steps of:

B. soaking clean cubilose powder to obtain cubilose pulp;

C. adding water into the cubilose pulp in a weight ratio of 1-5 times of the cubilose pulp, and continuously stirring for 3-8 hours at 70-120 ℃ at 200-1000 rpm to obtain a first extracting solution;

D. uniformly stirring the first extracting solution at the reduced temperature of 30-40 ℃, standing, removing a first lower layer liquid containing precipitates after impurities are precipitated, and taking out a supernatant as a second extracting solution;

E. stirring the rest liquid at 30-40 ℃ and standing, and removing a second lower layer liquid containing precipitates after further impurities are precipitated to obtain a third extracting solution;

F. and combining the second extracting solution and the third extracting solution to obtain the liquid cubilose extract.

According to one embodiment, in the step D, the mixture is stirred for 15 to 60 minutes at a stirring speed of 100 to 300rpm and then is allowed to stand for 3 to 4 hours.

According to one embodiment, in the step E, the mixture is stirred for 15 to 60 minutes at a stirring speed of 100 to 300rpm and then is allowed to stand for 2 to 3 hours.

According to a preferred embodiment, the stirring in step C is continued for 4 to 6 hours at a stirring speed of 300 to 800 rpm.

According to one embodiment, the step B comprises adding water in an amount of 15-40 times the weight of the clean cubilose powder, mixing, and soaking for 4-12 hours, preferably 6-10 hours. Preferably, the clean cubilose powder is mixed with water, then is subjected to ultrasonic crushing for 15-30 minutes and then is soaked. More preferably, the soaking is carried out at a temperature of 40 ℃ to 50 ℃.

The method of the invention also comprises the following steps: clean cubilose powder is obtained. In the present invention, clean bird's nest powder can be obtained by any suitable method. In general, the following steps may be included:

a) manually or mechanically pulverizing the bird's nest or bird's strip into bird's nest powder, and removing hair and foreign matters by hair suction, sieving and centrifugal separation to obtain clean bird's nest powder; or

b) The bird's nest powder is prepared by softening bird's nest or strips with water, manually removing hair and impurities, and manually or mechanically pulverizing.

According to an embodiment of the invention, the method may further comprise the step G: the liquid bird's nest extract is further processed to obtain a concentrated or solid bird's nest extract.

According to the invention, the solid cubilose extract can be obtained by drying. The method of drying is not particularly limited, and any suitable method may be used. Examples thereof include, but are not limited to, spray drying, vacuum drying, freeze drying and the like. The concentrated bird's nest extract can be obtained by a method such as distillation under reduced pressure, but is not limited thereto.

Preferably, the solid bird's nest extract is powder

The process of the invention further comprises a step H: and combining the first lower layer liquid and the second lower layer liquid to be used as fertilizer.

The method of the invention further comprises step I: and D, putting the hair and the impurities removed in the step A into a swallow house to attract swiftlet to come into contact to build the nest.

According to a second aspect of the present invention, there is provided a bird's nest extract obtained according to the above-described bird's nest extraction method.

According to a third aspect of the present invention, there is provided a bird's nest extract in which proteins having a molecular weight of 1000Da or less account for 45 wt% or more of the total amount of proteins in the extract, and the content of 8 kinds of human essential amino acids accounts for 45 wt% or more of the total amino acid content.

According to a preferred embodiment, the protein with a molecular weight below 3000Da in the cubilose extract accounts for more than 70 wt% of the total protein in the extract, and even can reach 75 wt%.

More preferably, the content of sialic acid in the cubilose extract accounts for more than 10 wt% of the total solid weight of the extract, and particularly preferably more than 12 wt% t, even more preferably more than 13 wt%.

According to a fourth aspect of the invention, a cubilose product is provided, which comprises the cubilose extract.

According to the present invention, the bird's nest product may be one of food, health products and cosmetics.

The food product may be a beverage.

The cubilose extracting method of the invention adopts the steps of stirring for a certain time at a certain temperature and stirring speed, and further standing, so that part of amide bonds in macromolecular proteins are broken, and an extract which is beneficial to human body absorption and has small molecular weight and concentration is formed. The method is mild, and no additional chemical substances (such as acid, enzyme, etc.) are added, so that the nutritional ingredients in the bird's nest are better retained. Analyzing the extract obtained by the method by HPLC (high performance liquid gel chromatography), wherein the content of small molecular peptides with molecular weight within 1000Da is more than 45 wt%; the amount of small molecular peptides and polypeptides with molecular weight within 3000Da can reach above about 75 wt%. The extract contains 18 amino acids, and according to a preferred embodiment, the weight ratio of 8 amino acids in the total 18 amino acids essential to human body is up to 48 wt%; sialic acid content, based on the total amount of extract (dry weight), is as high as 13.8 wt%, so that nutritional preservation and application maximization of the bird's nest are achieved in a targeted and effective manner; meanwhile, the method of the invention can obtain excellent extraction rate, and the weight ratio of the cubilose extract (dry weight) to the cubilose input in the preferred embodiment can reach more than 90 wt%.

Drawings

FIG. 1 is a flow diagram of a method for cubilose extraction according to one embodiment of the invention;

FIG. 2 is a histogram of the molecular weight distribution of protein peptides in the bird's nest extract obtained in example 3;

FIG. 3 is a photograph of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of molecular weight distributions of the bird's nest extracts obtained in example 3, comparative example 1 and comparative example 2, respectively.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following detailed description of the invention and the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

It should be noted that, in this document, the terms "comprises", "comprising" or any other variation thereof are intended to cover a non-exclusive inclusion, so that a method or apparatus including a series of elements includes not only the explicitly recited elements but also other elements not explicitly listed or inherent to the method or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other related elements in a method or apparatus that comprises the element.

It should be noted that the terms "first \ second" and "first \ second" are used herein only for distinguishing similar objects and do not represent a specific ordering for the objects, and it should be understood that "first \ second" and "first \ second" may be interchanged in a specific order or sequence where permitted. It should be understood that "first \ second" distinct objects may be interchanged under appropriate circumstances such that the methods of the present invention described herein may be performed in sequences other than those illustrated or described herein.

Generally, proteins can be classified into macromolecular proteins with molecular weight more than 10000Da and proteins with molecular weight less than 10000Da according to their molecular weight, and proteins with smaller molecular weight can be further subdivided into those with molecular weight of 3000Da to 10000Da which can be called long peptides, those with molecular weight between 1000Da to 3000Da which are often called polypeptides, and those with molecular weight less than 1000Da which are often called small peptides or oligopeptides. Since the extract obtained by the method of the invention contains polypeptides with molecular weight less than 3000Da up to about 75 wt%, and small peptides (or small peptides) with molecular weight less than 1000Da up to 45 wt%, the term "protein peptide" is used herein to refer to the polypeptides and small peptides in the nidus Collocaliae extract.

Hereinafter, the method for extracting bird's nest and its advantages according to an embodiment of the present invention will be described with reference to fig. 1. The obtained nidus Collocaliae material such as herba Swertiae Bimaculatae is first removed feather and impurities. Of course, the processed clean bird's nest material such as the bird's nest or the bird's strip can be directly used.

As shown in FIG. 1, firstly, step A is carried out to crush and remove impurities from the bird's nest material. Specifically, the bird's nest or strips with the feathers and impurities substantially removed can be firstly crushed into bird's nest powder at normal temperature by a hand or a grinding crusher. Because the unremoved feathers are lighter than the crushed bird's nest and the inorganic impurities such as dust, pebbles and the like are heavier after being crushed, fine and clean bird's nest powder is obtained after the hair and foreign matters in the bird's nest powder are removed in a hair sucking, sieving and centrifugal separation mode. Specifically, for example, a vibrating screen of 40 to 80 meshes can be used to obtain the bird's nest powder with uniform particle size, and the particles with larger particle size can be returned to the step A for re-crushing.

Alternatively, the bird's nest powder can be obtained by spraying clean water on the parts of the bird's nest or the bird's strips where the hairs and foreign matters exist, softening the bird's nest or the bird's strips, removing the hairs and the foreign matters by using tweezers, and grinding the bird's nest or the bird's strips. In the method, softened herba Swertiae Bimaculatae should be dried before grinding. Or pulverizing with heating pulverizer (temperature of 30-80 deg.C) to obtain dried nidus Collocaliae powder.

In the process of grinding the bird's nest into powder, whether the grinding is manual or machine grinding, the material should be prevented from becoming gel.

The method for removing the feathers and the impurities in the raw materials of the cubilose comprises any one of pure manual work, manual work and semi-automatic machinery and full-automatic cleaning mechanical hands. The present invention is not particularly limited thereto.

And step B, pretreating the cubilose powder, namely soaking the clean cubilose powder in water to expand the particles of the cubilose powder. Specifically, clean cubilose powder and water are mixed according to a certain proportion and soaked until the cubilose powder expands to obtain cubilose pulp. Preferably, the bird nest pulp can be obtained by soaking for 4-12 hours, preferably 6-10 hours after ultrasonication (for example, 40KHz frequency and 250W power can be used) for 15-30 minutes. The mixing ratio (weight ratio) of the cubilose powder and the water is 1:15 to 1: 40. The soaking step may also be carried out at a temperature, for example between 40 ℃ and 50 ℃, such that an optimal expansion of the powder of cubilose is obtained.

The water used in any step of the present invention may be one of purified water, deionized water or distilled water (e.g., double distilled water).

In the step C, water is added into the cubilose pulp obtained in the last step according to the weight ratio of the cubilose pulp to the water of 1:1 to 1:5, and the cubilose pulp is continuously stirred for 3 to 8 hours, preferably for 4 to 6 hours at the temperature of 70 to 120 ℃ and the rotating speed of 200 to 1000rpm, preferably 300 to 800 rpm. This step can cause the nutrients in the bird's nest to loosen and break part of the amide bonds of the peptide chains in at least part of the high molecular weight proteins, reducing the molecular weight of the proteins. This step yielded first extract GY 1.

And D, putting the first extracting solution GY1 into a mixing and stirring container, heating to 30-40 ℃, stirring at the rotating speed of 100-300 rpm for 15-60 minutes, for example 20-40 minutes, and standing. Stirring for a certain period of time helps to release impurities and to break the peptide bonds in the protein further loose into smaller molecular weight polypeptides. The standing time can be determined according to actual conditions. For example, the protein can be left for 3-4 hours to precipitate most impurities, and the protein can complete the reaction of breaking peptide bonds to generate the polypeptide with smaller molecular weight, and the broken polypeptide is dissolved in water. The lower precipitate containing secondary metabolites and gray matter was then discharged, and the supernatant was taken as second extract GY 2.

In step E, the remainder of the mixing and stirring vessel, i.e., the middle layer, is also stirred at 30-40 ℃ for 15-60 minutes, for example, 20-40 minutes at 100-300 rpm, and then left to stand. The standing time can be 2-3 hours, so that impurities can be fully precipitated. The lower precipitate was also drained off and the supernatant (usually all remaining liquid in this step) was extracted as third extract GY 3.

In step F, the obtained second extracting solution GY2 and the third extracting solution GY3 are combined to form a fourth extracting solution GY4, namely a liquid bird's nest extract.

Still referring to fig. 1, the liquid bird's nest extract obtained in step F may be further subjected to step G to obtain a concentrated or solid extract. For example, the fourth extract GY4 can be put into a spray dryer with an automatic feeding system and spray-dried to obtain a powdery solid. The concentrated/solid bird's nest extract can also be obtained by methods such as lyophilization, distillation under reduced pressure, vacuum drying, and the like. The form of the bird's nest extract is not particularly limited in the present invention, and the further treatment mode can be selected as required.

The effective utilization rate (namely extraction rate) of the cubilose components in the cubilose extract obtained by the method reaches more than 90 percent. The proteins in the extract are mostly present as polypeptides with a lower molecular weight. The molecular weight of the protein and the polypeptide is in narrow normal distribution, wherein the content of the polypeptide with lower molecular weight (less than 1000Da) accounts for more than 45 wt%, and the content of the polypeptide with molecular weight less than 3000Da can reach about 75 wt%. This narrow normal distribution of small molecular weight polypeptides allows for better utilization efficiency. The extract obtained by the method contains 18 amino acids, and the content proportion of 8 amino acids necessary for human bodies is more than 45 wt%, even can reach 48 wt%; the sialic acid content in the extract (dry weight) is above 10 wt%, up to 13.8 wt%. Compared with other extraction methods, the method has remarkable advantages in nutrition and value components.

The cubilose contains protein with high proportion, but the protein has large molecular weight (which can be 10000 Da-300000 Da, even higher) and is not easy to absorb due to the three-stage supercoiled structure. Even for polypeptides with a molecular weight of about 3000Da, the human absorption efficiency is not high (about 40%). Small molecule short peptide (molecular weight below 1500 Da) with molecular weight between polypeptide and amino acid can be directly absorbed through intestinal epidermis, and enter blood to be delivered to required parts of human body, and still can maintain the nutritional components and excellent biological activity and physiological function. The extract of the invention is mostly short peptide, the proportion is far higher than that of the cubilose product obtained by the conventional method, and the extract is more beneficial to the absorption and utilization of the human body.

The method of the present invention may further comprise steps H and I for recycling the accumulated waste in the method. Step H involves using the lower precipitate (secondary metabolite and gray matter) obtained in steps D and E above as a biodegradable material to be used as a fertilizer. Step I comprises the steps of reusing inedible feather and impurities in the swallow house to attract more swallows with taste to build the nest. The method realizes zero waste and zero emission in waste management to reduce waste treatment of utilizing energy and generating methane and carbon dioxide through product gas, and effectively protects the global environment.

The cubilose extract can be further formed into products such as food, health products, beauty cosmetics and the like. The food or health care product of the invention contains the cubilose extract in any form. The product may also contain other components such as additives, adjuvants, excipients, etc. Wherein the additive may be such as a pigment, a flavoring agent, a fragrance, a sweetener, etc. The product may also contain further active ingredients, such as other nutritional ingredients, which may be other nutritional ingredients that are complementary to or synergistic with the nutrients in the bird's nest.

The advantages of the invention are further illustrated by the following specific examples.

Examples

The analysis method comprises the following steps:

1. measurement of protein molecular weight and molecular weight distribution: the extract samples obtained in the following examples and comparative examples were analyzed for molecular weight and distribution thereof by high performance liquid gel chromatography (Agilent 1300series, HPLC). The specific analysis conditions were as follows: the chromatographic column adopts a TSKgelG2000SWXL gel chromatographic column, the molecular weight classification range is 100-20000, the detection wavelength of a diode array detector is 220nm, and the column temperature is 30 ℃; the mobile phase was ethyl/water/trifluoroacetic acid (45/55/0.1 (v/v/v)), the flow rate was 0.5 mL/min, and the sample size was 5-20 μ l. The results are shown in tables 1 to 4.

2. SDS-PAGE: in order to more intuitively compare the distribution of the molecular weight of the protein in the extract obtained by the method of the present invention with that of the extract obtained by the prior art, the distribution of the molecular weight of the protein in the extract sample was determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

Analytical instruments and materials: FD-201 electrophoresis apparatus (from Shanghai medical analytical Instrument factory), small vertical electrophoresis accessory mold and TEMED (from BIO-RAD), peptide molecular mass standard Mr range 348-.

The specific analysis method comprises the following steps: the cathode buffer is filled in the inner tank of the electrophoresis apparatus, the anode buffer is used in the outer tank, electrophoresis is carried out at constant voltage, electricity is supplied for 1 hour under 40V, when the sample enters the separation gel, the voltage is raised to 60V, and the electrophoresis is carried out for 2 hours. After electrophoresis, the gel was placed in a staining solution [ 25g/L Coomassie Brilliant blue, R250 in ethanol (V): glacial acetic acid (V): oscillating and dyeing in water (V) at a ratio of 9:2: 9) for 1.5-2 hours, transferring into a decoloring solution (400ml/L ethanol and 40ml/L glacial acetic acid) for diffusion decoloring until the background is clear. And (4) taking a picture to obtain a more visual gel electrophoresis picture of the molecular weight distribution of the extract sample, which is shown in figure 3.

3. And (3) determining the types and the contents of the total amino acids in the extract: according to GB/T5009.124-2003 "determination of amino acids in food", an amino acid autoanalyzer (Hitachi L-8900 amino acid autoanalyzer) was used to hydrolyze an extract test sample into free amino acids by hydrochloric acid, and the free amino acids were separated by an ion exchange column of the amino acid autoanalyzer and subjected to color reaction with ninhydrin solvent (Japan and Wako pure chemical industries, Ltd.) to determine the kinds and contents of amino acids in the bird's nest extract of examples of the present invention and the comparative example. The specification of the chromatographic column is 4.6 multiplied by 60mm, and the filler is a sulfonic acid type cation resin separation column with the diameter of 3 mu m.

The specific analysis method comprises the following steps: 1) sample pretreatment: about 0.5g (to the accuracy of 0.0001g) of each of the extracts GY4-1, GY4-2, GY4-3 of the following examples 1, 2 and 3 and the extracts of comparative examples 1 and 2 was sampled in a hydrolysis tube, 15mL of 6mol/L hydrochloric acid was added, 3 drops of freshly distilled phenol were added, the tube was frozen in a refrigerant for 5 minutes, and then vacuum (approximately 0Pa) was applied, high purity nitrogen was applied, vacuum was applied, nitrogen was applied, 3 times were repeated, the tube was sealed under nitrogen application, the sealed tube was placed in a thermostatic drying oven at 110. + -. 1 ℃ for hydrolysis for 22 hours, and then taken out and cooled. And opening the hydrolysis tube, filtering the hydrolysate, washing the hydrolysate with ionized water for multiple times, transferring the hydrolysate to a 50mL volumetric flask, and metering the volume with the deionized water. 2mL of the filtrate was aspirated into a 50mL beaker, evaporated to dryness in a water bath, and the residue was dissolved in 1-2mL of water and dried again and repeated twice. Finally, after evaporation to dryness, the mixture is dissolved by 10mL of buffer solution with pH2.2, and the mixture is evenly mixed and filtered for determination. 2) Quantitative analysis: 0.200mL of each standard amino acid solution of 18 different amino acids (from Fluka AAS18) was accurately pipetted and diluted to 5mL with pH2.2 buffer, the standard dilution having a concentration of 2.0 nmol/20. mu.L, as the amino acid standard for the on-machine determination. The amino acid content and corresponding content of each extract sample were determined by a external standard method using an amino acid autoanalyzer. The results are shown in Table 5.

4. Determination of sialic acid content: sialic acid content tests were performed on samples of the extracts of the examples and comparative examples and on commercially available canned swiftlet nest using high performance liquid chromatography (Waters2695 and Waters2489 uv detectors).Sialic acid control (purity 99% or more) was purchased from Sigma and the column was Waters Atlantis T₃(4.6 mm. times.150 mm,5 μm) and the mobile phase acetonitrile-water (40: 60). Weighing 100mg of each extract sample, placing in an erlenmeyer flask, accurately adding 60mL of 1.0% phosphoric acid solution, adding 2mL of ninhydrin indicator, hydrolyzing in 100 deg.C boiling water bath for 20 min, cooling to room temperature, supplementing weight loss, shaking, and filtering with 0.45 μm filter membrane. The detection flow rate is 1.0mL/min, the detection wavelength is 192nm, and the injection volume is 20 muL. The results are shown in Table 6.

Example 1

800g of bird's nest raw material (Swiftlet Garden, Dissauu, Mo.). Removing feather and impurities in the raw material of the cubilose, and crushing the cubilose or cubilose strips into cubilose powder by a grinding crusher at normal temperature. The fine and clean cubilose powder of 680g is obtained after the hair and foreign matters on the cubilose are removed by hair suction, filtration and centrifugal separation. To avoid gelling of the material due to shear heat, the mill-crusher chamber temperature was set at 30 ℃.

Putting the crushed bird's nest powder into purified water according to the weight ratio of 1:20, carrying out ultrasonic crushing for 20 minutes by an ultrasonic vibrator (the ultrasonic frequency is 40KHz, the ultrasonic power is 250W), heating to 50 ℃, soaking for 6 hours, and expanding the bird's nest powder particles to the optimum value to obtain the bird's nest slurry GY0-1 of 14280g in total for effective extraction.

Taking GY0-1 out, adding purified water at a weight ratio of 1:4, stirring at 500rpm, heating at 120 deg.C for 4 hr to loosen nutrients in nidus Collocaliae, to obtain extract GY1-1 of 71400 g.

Placing the extract GY1-1 in a container with a stirrer, heating to 30 deg.C and stirring at 300rpm for 30 minutes, standing for 3 hours to remove the lower layer precipitate to obtain supernatant GY2-1 and 13138 g.

After heating the residue in the vessel to 40 ℃ and stirring at 100rpm for 30 minutes, the lower layer of precipitate was removed by standing for 2 hours to obtain 52550g in total of GY3-1 as a supernatant.

Mixing the obtained supernatant GY2-1 and supernatant GY3-1 to obtain 65688g of extracting solution GY 4-1.

The weight ratio and number average molecular weight of each protein in the extractive solution containing protein peptide obtained by the above analysis method are shown in Table 1 below

Table 1: example 1 weight percent and number average molecular weight of protein containing protein peptide in extract of bird's nest

Molecular weight Range (Da)	Weight percent (wt%, λ 220nm)	Number average molecular weight
			＜180	1.43
180～500	16.97	380
			501～1000	27.61	641
1001～2000	19.23	1313
			2001～3000	8.77	2416
3001～5000	6.96	3709
			5001～8000	4.89	5616
8001～10000	4.28	8197
			＞10000	9.86	18031

As can be seen from Table 1, the proportion of small peptides with a molecular weight of < 1000Da is about 46 wt%, and the proportion of protein peptides (including small peptides and polypeptides, the same below) with a molecular weight of <3000Da is up to 74 wt%.

The extract was passed through a spray dryer and spray-dried to obtain 626g of powdery bird's nest extract. The extraction rate of the obtained bird's nest component (the final extract accounts for the weight ratio of the input bird's nest) relative to the weight of the bird's nest material obtained in the embodiment reaches 92 percent.

Example 2

800g of raw material of bird's nest (Swiftlet Garden, Dissaurus, Roffy, Malaya) was treated in the same manner as in example 1 to obtain 710g of bird's nest powder. Adding the crushed bird's nest powder into distilled water according to the weight ratio of 1:30, carrying out ultrasonic crushing for 20 minutes by an ultrasonic vibrator (the ultrasonic frequency is 40KHz, the ultrasonic power is 250W), and soaking for 8 hours at 40 ℃ to ensure that the bird's nest powder particles are expanded to be optimal. A total of 22010g of cubilose slurry GY0-2 is obtained to be effectively extracted.

Taking GY0-2 out, adding into distilled water at a weight ratio of 1:3, stirring at 300rpm, heating at 80 deg.C for 5 hr to loosen nutrients in nidus Collocaliae, and extracting to obtain extract GY1-2 of 88040g in total.

Placing the extract GY1-2 in a container with stirring, heating to 40 deg.C and stirring at 200rpm for 30 min, standing for 4 hr to remove the precipitate, and collecting the supernatant GY2-2 to obtain 17520 g.

After the residue in the vessel was stirred at 100rpm for 30 minutes at 30 ℃, the vessel was left to stand for 2 hours to remove the lower layer precipitate to obtain 62116g of a total of GY3-2 as a supernatant.

The obtained supernatant GY2-2 and supernatant GY3-2 were mixed to obtain 79636g of an extract GY 4-2.

The weight ratio and the number average molecular weight of the protein peptide contained in the extract GY4-2 in each molecular weight range were measured as shown in Table 2 below:

table 2: example 2 weight percent protein and number average molecular weight of protein peptide contained in extract solution of bird's nest

Molecular weight Range (Da)	Weight percent (wt%, λ 220nm)	Number average molecular weight
			＜180	1.58
180～500	18.12	389
			501～1000	29.22	703
1001～2000	19.63	1314
			2001～3000	9.11	2411
3001～5000	6.12	4021
			5001～8000	4.29	5333
8001～10000	3.76	8461
			＞10000	8.17	16976

Wherein, the proportion of the small molecular peptide with the molecular weight less than 1000Da is about 48.9 wt%, and the proportion of the protein peptide with the molecular weight less than 3000Da is 77.7 wt%.

The extract GY4-2 was put into a spray dryer equipped with an automatic feeding system and spray-dried to obtain 642g of a powdery bird's nest extract. The extraction rate of the obtained bird's nest component (the final extract accounts for the weight ratio of the input bird's nest) relative to the weight of the bird's nest material is up to 90.4 percent.

Example 3

800g of bird's nest raw material (Swiftlet Garden, Dissauu, Mo.) was used to obtain 701g of bird's nest powder according to the method of example 1. Soaking the crushed cubilose powder in clean water for 10 hours in a weight ratio of 1:40, carrying out ultrasonic crushing for 20 minutes by an auxiliary ultrasonic vibrator (ultrasonic frequency is 40KHz, ultrasonic power is 250W), and preferably heating to 40 ℃ to ensure that the cubilose powder particles are expanded to the best. The bird's nest slurry GY0-3 which obtains the maximum expansion effect of the bird's nest powder particles totally 28741g for effective extraction.

Taking out GYO-3, adding distilled water at a weight ratio of 1:2, stirring at 200rpm, heating to 75 deg.C for 6 hr to loosen nutrients in nidus Collocaliae, and extracting to obtain total 42840g of extract GY 1-3.

Placing the extract GY1-3 into a container with stirring, heating to 40 deg.C and stirring at 100rpm for 30 minutes, standing for 3 hours to remove the lower layer precipitate to obtain supernatant GY2-3 of 8567 g.

After the residue in the vessel was stirred at 40 ℃ for 30 minutes at 100rpm, it was left to stand for 3 hours to drain the lower layer precipitate, and 30417g in total of GY3-3 was obtained as a supernatant.

The obtained supernatant GY2-3 and the supernatant GY3-3 were mixed to obtain an extract GY4-3 of 38984 g.

The weight ratio and number average molecular weight of the protein peptide contained in the extract GY4-3 were determined by the above method and are shown in Table 3 below and FIG. 2:

table 3: example 3 weight percent protein and number average molecular weight of protein peptides contained in bird's nest extract

Wherein, the proportion of the small molecular peptide with the molecular weight less than 1000Da is about 48.9 wt%, and the proportion of the protein peptide with the molecular weight less than 3000Da is 77.7 wt%. It can be seen more intuitively from FIG. 2 that the molecular weight of the protein peptide in the extract obtained by the method of the present invention is in a narrow normal distribution.

The extract GY4-3 was put into a spray dryer equipped with an automatic feeding system and spray-dried to obtain 638g of powdery bird's nest extract. The extraction rate of the obtained bird's nest component (the final extract accounts for the weight ratio of the input bird's nest) of the dry extract obtained in the embodiment is 91 percent relative to the weight of the bird's nest material.

Comparative example 1

This comparative example used an enzymatic method to extract the bird's nest.

Taking cubilose raw material (Swiftlet Garden of Dissaulu biological valley, Roffia, Malaysia), crushing, removing impurities, cleaning, screening by a 40/80-mesh filter screen to obtain 10g of cubilose powder, adding water according to the weight ratio of 1:60, soaking for 4 hours at room temperature, placing in a closed container, heating in a 90 ℃ water bath for 30 minutes, and then cooling to 50 ℃. Stir at 3000rpm for 10 minutes. Dilute hydrochloric acid was added to the homogenized bird's nest to adjust pH to 7, and then 13mg of papain (20 ten thousand U/g enzyme activity, available from Nanning Pombo bioengineering Co., Ltd.) and 13mg of alkaline protease (20 ten thousand U/g enzyme activity, available from Nanning Pombo bioengineering Co., Ltd.) were added and reacted at 50 ℃ for 3 hours in a constant temperature shaking table. Heating the enzymolysis product to 95 deg.C, and inactivating enzyme for 30 min. Then, filtering with 200 mesh filter cloth to remove impurities to obtain clear enzymolysis solution. Finally, 6.8 g of powdery cubilose extract is obtained by spray drying. The extraction rate was 68%.

High Performance Liquid Chromatography (HPLC) detection shows that the obtained nidus Collocaliae extract contains small molecular peptide with molecular weight less than 1000Da 31 wt%, and small molecular peptide and polypeptide below 3000Da 47 wt%. Sialic acid content detected by spectrophotometry on sialic acid samples was 9.51%. The content of Essential Amino Acids (EAA) in the extract in total amino acids is 39.27 wt% by ninhydrin color development.

Comparative example 2

The comparative example adopts the traditional preparation method of cooking with water, steaming and stewing to extract the cubilose

Taking cubilose raw material (swift Garden of Dishalu biological grain, Roffia, Malaysia), crushing, removing impurities, cleaning, screening by a 40/80-mesh filter screen to obtain 10g of cubilose powder, mixing with water according to the weight ratio of 1:10, and soaking for 4 hours at room temperature. Placing the soaked nidus Collocaliae in a sealed container, stewing at 90 deg.C for 6 hr, cooling, filtering with 200 mesh filter membrane, and concentrating. Spray drying to obtain 1.42 g of powdery extract, with extraction rate of only 14.2%. Through high performance liquid gel chromatography (HPLC) detection, the content ratio (area under the curve) of the small molecular peptides with the molecular weight of less than 1000Da in the obtained cubilose is 14%, and the content (area under the curve) of the small molecular peptides and polypeptides with the molecular weight of less than 3000Da is 23%. Sialic acid content was 9% as detected by spectrophotometry on sialic acid samples. The content of Essential Amino Acids (EAA) in the extract in total amino acids is 39.29% by ninhydrin color development.

And (3) testing results:

1. the results of measuring the molecular weight distributions of the protein and polypeptide in the extract obtained by the enzymatic extraction method in comparative example 1, the extract obtained by the boiling and steaming method in comparative example 2 and the extract of example 3 by the above-mentioned high performance liquid gel chromatography (HPLC) are shown in Table 4 below.

Table 4: molecular weight distribution of proteins/polypeptides in extracts of example 3, comparative example 1 and comparative example 2

From table 4, it can be seen that, compared with other methods for extracting edible bird's nest, the molecular weight of protein peptide in edible bird's nest extract obtained by the extraction method of the present invention is not only small (<3000Da), but also the distribution is uniform and concentrated, and the protein peptide has a more obvious normal distribution characteristic.

Further referring to FIG. 3, SDS-PAGE photographs of molecular weight distributions of protein peptides in the extract obtained by the method of the present invention (example 3), the enzymatic method (comparative example 1) and the poached and stewed method (comparative example 2) are shown corresponding to Table 4. FIG. 3 is a photograph 1 of a gel electrophoresis of protein peptides extracted from an extract liquid obtained by the extraction method of the present invention, in which the molecular weight distribution of the protein peptides is narrow and a high proportion of the protein peptides is distributed in a region of less than 3000 Da. Photograph 2 shows the molecular weight distribution of the protein in the extract obtained by the enzymolysis method of comparative example 1, although the small molecular peptides and the polypeptides have a certain ratio, the distribution of each molecular weight is scattered, and the ratio in the interval of 3000-10000Da is significantly higher than that in example 3, reaching 44%. Photograph 3 shows the molecular weight distribution of the protein in the extract obtained by the steam boiling and stewing method of comparative example 2, which is not shown in the picture because the amount of the small peptides and polypeptides obtained is small and most of them are distributed in the region with molecular weight more than 10000 Da. It can be seen that although the enzymolysis method can obtain peptides with smaller molecular weight, the distribution of molecular weight is not concentrated, and a larger proportion of proteins with larger molecular weight and more dispersed distribution still exist, which affects the specificity and stability of the product efficacy. However, the traditional water boiling, steaming and stewing method is difficult to obtain the peptide with smaller molecular weight, and most of the protein in the extract still has very high molecular weight, so that the absorption of the nutritional value is influenced.

2. The amino acid components and content ratios of the extract samples of examples 1 to 3 and comparative examples 1 to 2 were measured by an automatic amino acid analyzer using the High Performance Liquid Chromatography (HPLC) principle as described above are shown in Table 5.

The comparative results in table 5 below show: compared with the extracts obtained by the cubilose enzymolysis extraction method and the traditional hydrolysis steaming cubilose extraction method, the cubilose extract obtained by the extraction method contains 18 amino acids, the weight content of 8 human Essential Amino Acids (EAA) reaches about 48 wt%, and is obviously higher than the weight ratio of the human essential amino acids (39.27 wt% and 39.29 wt% respectively) in the extracting solution obtained by the comparative example 1 and the comparative example 2. This demonstrates that the protein in the bird's nest is better retained by the method of the invention. According to the invention, after the bird's-nest is crushed, impurities are removed by hair sucking, sieving and centrifuging, so that water washing is avoided, and more soluble protein is retained.

Table 5: examples 1-3, comparative examples 1 and 2, the content of amino acids in the extract liquid

3. Sialic acid in the bird's nest extract in examples 1-3, comparative examples 1 and 2, and commercially available rock candy bird's nest samples was detected spectrophotometrically according to the above detection method, and the results are shown in table 6 below after 6 times of detection for each sample.

Table 6: content of sialic acid in extracts and commercially available products of examples 1 to 3, comparative example 1 and comparative example 2

	Example 1	Example 2	Example 3	Comparative example 1	Comparative example 2	Commercially available product
							Sialic acid (wt%)	13.82	13.81	13.85	9.51	9.08	1.04

The results of the above table show: the average content of sialic acid in the cubilose extracting solution obtained by the extraction method of the invention is up to 13.82 wt%, which is far higher than 9.51 wt% and 9.08 wt% in the cubilose extracting solution obtained by an enzymolysis method and a water cooking and steaming method and 1.04 wt% of the cubilose extracting solution sold in the market.

4. The extraction rates according to examples 1, 2 and 3 of the present invention and comparative examples 1 and 2 were compared, and the extraction rate (%) was expressed in table 7 as dry weight of extract/weight of bird's nest input.

Table 7: comparison of extraction yields of examples 1 to 3, comparative example 1, and comparative example 2

	Example 1	Example 2	Example 3	Comparative example 1	Comparative example 2
						Extraction ratio (wt%)	92	90.4	91	68	14.2

The above results show that: the content proportion of the cubilose extract obtained by the extraction method of the invention reaches more than 90 wt%, and the extraction efficiency is far higher than the extraction efficiency (68 wt% and 14.2 wt%) obtained by an enzymolysis method and a water boiling, steaming and stewing method.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents made by the contents of the present specification and drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A method for extracting bird's nest, the method comprises the following steps:

B. soaking clean cubilose powder to obtain cubilose pulp;

E. d, continuously stirring the residual liquid in the step D at 30-40 ℃, standing, and removing a second lower layer liquid containing precipitates after further impurities are precipitated to obtain a third extracting solution; and

2. The method according to claim 1, wherein the stirring speed in step D is 100-300 rpm for 15-60 minutes, and the mixture is allowed to stand for 3-4 hours.

3. The method according to claim 1, wherein in the step E, the mixture is stirred for 15 to 60 minutes at a stirring speed of 100 to 300rpm and then is allowed to stand for 2 to 3 hours.

4. The method according to claim 1, wherein the stirring in step C is continued at a stirring speed of 300 to 800rpm for 4 to 6 hours.

5. The method according to claim 1, wherein the step B comprises adding water in an amount of 15-40 times the weight of the clean cubilose powder, mixing and soaking for 4-12 hours, preferably 6-10 hours.

6. The method according to claim 5, wherein the clean cubilose powder is mixed with water, then is subjected to ultrasonic crushing for 15-30 minutes, and then is subjected to soaking, and preferably the soaking is performed at a temperature of 40-50 ℃.

7. The method of claim 1, wherein the method further comprises the steps of:

A. obtaining the clean cubilose powder, which comprises the following steps:

8. The method of claim 1, wherein the method further comprises the steps of:

G. the liquid bird's nest extract is further processed to obtain a concentrated or solid bird's nest extract.

9. The method of claim 1, wherein the method further comprises the steps of:

H. and combining the first lower layer liquid and the second lower layer liquid to be used as fertilizer.

10. The method of claim 7, wherein the method further comprises the steps of:

I. and D, putting the hair and the impurities removed in the step A into a swallow house to attract swiftlet to come into contact to build the nest.

11. A bird's nest extract obtained by the method for extracting bird's nest according to any one of claims 1-10.

12. A nidus Collocaliae extract contains protein with molecular weight below 1000Da 45 wt% of total protein content, and 8 essential amino acids 45 wt% of total amino acids; preferably, the protein with molecular weight below 3000Da in the cubilose extract accounts for more than 70 wt% of the total protein in the extract; more preferably, the content of sialic acid in the cubilose extract accounts for more than 10 wt% of the total solid weight of the extract.

13. A bird's nest product comprising the bird's nest extract of claim 11 or 12, preferably the bird's nest product is one of a food, a health product, and a cosmetic product, more preferably the food is a beverage.