Method for extracting high-purity cubilose polysaccharide by combining green high-energy physical field and application

Abstract

The invention discloses a method for extracting high-purity cubilose polysaccharide by combining a green high-energy physical field and application. The method for extracting the cubilose polysaccharide comprises the following steps: mixing cubilose with water, and performing ultrahigh pressure treatment and dispersion to prepare cubilose homogenate; performing pulsed electric field treatment on the bird's nest homogenate, performing solid-liquid separation, and collecting supernate; and filtering the supernatant, collecting the filtrate, and drying to prepare the cubilose polysaccharide. According to the method for extracting the cubilose polysaccharide, the polysaccharide is extracted from the cubilose through ultrahigh-pressure treatment, high-speed dispersion, pulsed electric field and other high-energy physical field combined treatment, no organic reagent is added in the operation process, the operation is carried out under the room temperature condition, the technological process is simple and easy to implement, no reagent except purified water is used in the method, and the method is environmentally friendly. The method is a green preparation method.

Description

Technical Field

[0001] This application belongs to the technical field of food processing, and relates to a method and application for jointly extracting high-purity bird's nest polysaccharide by a green high-energy physical field. Further, it relates to a method and application for jointly extracting high-purity bird's nest polysaccharide with free radical scavenging, cell repair, and intestinal flora health effects by a green high-energy physical field, and can be used to prepare high-purity bird's nest polysaccharide with free radical scavenging, cell repair, and intestinal flora health effects. Background Art

[0002] Bird's nest contains rich proteins, polysaccharides, and sialic acids. Modern medical research shows that bird's nest has effects such as anti-inflammatory, promoting nerve development, enhancing immunity, antiviral, and beauty care. Among them, the bird's nest polysaccharide has been proven to have anti-inflammatory, antibacterial, and intestinal flora improvement effects. Sialic acid connected to the end of the sugar chain, scientifically named "N-acetylneuraminic acid", is a derivative of a nine-carbon monosaccharide, and it has been widely studied and proven to play an important role in nerve development. Thus, the polysaccharide in bird's nest is a high-value-added product of bird's nest with multiple effects.

[0003] The main steps of the current method for extracting bird's nest polysaccharide are: enzymatic hydrolysis, ammonium sulfate precipitation, desalting, secondary enzymatic hydrolysis and acid hydrolysis, two column chromatographies, and alcohol precipitation. This extraction method has low purity.

[0004] Therefore, there is an urgent need for a method to extract high-purity polysaccharide from bird's nest, which is of great significance for expanding the application of bird's nest. Summary of the Invention

[0005] Based on this, it is necessary to provide a method and application for jointly extracting high-purity bird's nest polysaccharide by a green high-energy physical field.

[0006] In some embodiments, a method for extracting bird's nest polysaccharide is provided, including the following steps:

[0007] Mix bird's nest with water, perform ultra-high pressure treatment, and disperse to prepare bird's nest homogenate;

[0008] Perform pulsed electric field treatment on the bird's nest homogenate, separate solid from liquid, and collect the supernatant; and,

[0009] Filter the supernatant, collect the filtrate and dry it to prepare bird's nest polysaccharide.

[0010] In some embodiments, the method for extracting bird's nest polysaccharide provided satisfies one or more of the following conditions (1) to (5):

[0011] (1) The mass ratio of the bird's nest to the water is 1:(20 - 70);

[0012] (2) The pressure of the ultra-high pressure treatment is 200 MPa to 500 MPa;

[0013] (3) The time of ultra-high pressure treatment is 5 min to 15 min;

[0014] (4) The rotation speed of dispersion is 8000 rpm to 12000 rpm; and,

[0015] (5) The time of dispersion is 1 min to 3 min.

[0016] In some embodiments, the method for extracting edible bird's nest polysaccharide provided satisfies one or more of the following conditions (1) to (4):

[0017] (1) The electric field strength of pulsed electric field treatment is 10 kV to 30 kV;

[0018] (2) The number of pulses of pulsed electric field treatment is 3 to 10;

[0019] (3) The pulse width of pulsed electric field treatment is 10 μs to 50 μs; and,

[0020] (4) The condition of solid-liquid separation is centrifugation at 8000 rpm to 10000 rpm for 20 min to 30 min.

[0021] In some embodiments, in the method for extracting edible bird's nest polysaccharide provided, performing the filtration includes microfiltrating the supernatant, collecting the first filtrate, and ultrafiltrating the first filtrate.

[0022] In some embodiments, in the method for extracting edible bird's nest polysaccharide provided, the pore diameter of the microfiltration membrane is 4 μm to 6 μm.

[0023] In some embodiments, the method for extracting edible bird's nest polysaccharide provided satisfies one or more of the following conditions (1) to (3):

[0024] (1) During ultrafiltration, the cut-off molecular weight is 40 kDa to 50 kDa;

[0025] (2) The centrifugation speed of ultrafiltration is 4000 rpm to 6000 rpm; and,

[0026] (3) The time of ultrafiltration is 20 min to 30 min.

[0027] In some embodiments, a bird's nest polysaccharide is provided. The monosaccharide composition of the bird's nest polysaccharide includes the following components: The monosaccharide composition of the bird's nest polysaccharide includes N-acetylneuraminic acid, mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Among them, the N-acetylneuraminic acid accounts for 31% - 32% of the total mass of the bird's nest polysaccharide, the mannose accounts for 7% - 8% of the total mass of the bird's nest polysaccharide, the fucose accounts for 1% - 2% of the total mass of the bird's nest polysaccharide, the galactose accounts for 25% - 26% of the total mass of the bird's nest polysaccharide, the N-acetylglucosamine accounts for 18% - 19% of the total mass of the bird's nest polysaccharide, and the N-acetylgalactosamine accounts for 11% - 12% of the total mass of the bird's nest polysaccharide.

[0028] In some embodiments, the bird's nest polysaccharide is prepared by the method described above.

[0029] In some embodiments, there is provided an application of the bird's nest polysaccharide prepared by the method described above or the bird's nest polysaccharide in at least one of the products for scavenging free radicals, improving intestinal flora, and repairing cells.

[0030] In some embodiments, in the provided application, the products include one or more of food, medicine, and skin care products.

[0031] The aforementioned method for extracting bird's nest polysaccharide uses combined treatment of high-energy physical fields such as ultra-high pressure treatment, dispersion, and pulsed electric field to extract polysaccharide from bird's nest. The purity of the extracted bird's nest polysaccharide is as high as 95.8%, and it has excellent antioxidant activity, antibacterial effect, and the function of improving intestinal flora, which is beneficial to the application of bird's nest polysaccharide in food, medicine, and skin care products.

[0032] In addition, no organic reagents need to be added during the operation process of this application. It is a green preparation method and can be operated at room temperature. The process flow is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to more completely understand this application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or examples. Obviously, the following drawings are only some embodiments of this application. For those skilled in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 It is the gas chromatogram of the monosaccharide composition of the bird's nest polysaccharide obtained in Example 1. Among them, Fuc is fucose; Man is mannose; Gal is galactose; GalNAc is N-acetylgalactosamine; GluNAc is N-acetylglucosamine;

[0035] Figure 2 Liquid chromatogram of N-acetylneuraminic acid in the edible bird's nest polysaccharide obtained in Example 1;

[0036] Figure 3 Scavenging ability of the edible bird's nest polysaccharide obtained in Example 1 against DPPH free radicals;

[0037] Figure 4 Repair effect diagram of the edible bird's nest polysaccharide on HaCaT cells;

[0038] Figure 5 PCoA analysis diagram, where A is the blank control group and B is the edible bird's nest polysaccharide group;

[0039] Figure 6 Analysis of differences in species composition at the family level, where A is the blank control group, B is the edible bird's nest polysaccharide group, Acidaminococcaceae is Acidaminococcaceae; Bacteroidaceae is Bacteroidaceae; Enterobacteriaceae is Enterobacteriaceae; Prevotellaceae is Prevotellaceae; Selenomonadaceae is Selenomonadaceae. Detailed implementation manners

[0040] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] It should be noted that when an element is referred to as "fixed to" another element, it can be directly on the other element or there may also be an intermediate element. When an element is considered to be "connected" to another element, it can be directly connected to the other element or there may be an intermediate element at the same time.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0043] Term

[0044] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0045] The terms “and / or”, “or / and”, and “and / or” used in this application cover any one of two or more related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more than two related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from “and / or”, “or / and”, and “and / or”, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by “logical AND” and also undoubtedly includes the technical solution connected by “logical OR”. For example, “A and / or B” includes three parallel solutions: A, B, and “the combination of A and B”.

[0046] In this application, the terms “a plurality of”, “multiple types of”, “multiple times”, “multiple elements”, etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, “one or more types” means one type or two or more types.

[0047] In this application, “its combination”, “any combination thereof”, “any combination mode thereof”, etc. include all suitable combination modes of any two or more of the listed items.

[0048] In this application, the “suitable” in “suitable combination mode”, “suitable mode”, “any suitable mode”, etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0049] In this application, “preferred”, “better”, “more preferable”, “it is advisable” are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0050] In this application, “furthermore”, “even further”, “especially”, etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of this application.

[0051] In this application, “optionally”, “optional”, “optional” mean that it can be either present or absent, that is, it refers to any one of the two parallel solutions of “present” or “absent”. If “optional” appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each “optional” is independent.

[0052] In the present invention, in relation to "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0053] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0054] In this application, regarding the numerical interval (i.e., numerical range), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints of this numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions for features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0055] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0056] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0057] "Room temperature" in this application generally refers to 5°C to 30°C, and preferably refers to 25 ± 5°C.

[0058] Currently, the methods for extracting polysaccharides mainly target plant polysaccharides. Plant polysaccharides mainly exist in cells. They can release polysaccharides by physically treating to break the cell wall or by acidolysis or enzymatic hydrolysis to degrade the cell wall. Some polysaccharides are also combined with proteins, but polysaccharides are the main component and the proportion of impurities is low. However, the polysaccharides in bird's nest are different. Bird's nest polysaccharides belong to animal polysaccharides, which are mostly combined with proteins and exist in the form of glycoproteins, and proteins are the main component. From the perspective of polysaccharide extraction, the proportion of impurities is high. This also increases the difficulty of extracting polysaccharides from bird's nest.

[0059] The steps for extracting polysaccharides from substances with high protein content are rather cumbersome. Currently, the commonly used methods include enzymatic hydrolysis method and alkaline extraction method. The main steps of the enzymatic hydrolysis method are: enzymatic hydrolysis, protein removal by Sevag method, desalting, gel column separation, alcohol precipitation, etc. Although enzymatic hydrolysis changes proteins from macromolecules to small molecules and releases some polysaccharides, the small molecule proteins after enzymatic hydrolysis have molecular weights close to some of the polysaccharides, making it more difficult to separate proteins and polysaccharides based on molecular weight differences, resulting in a still high protein content in the final product, that is, a low purity of polysaccharides. Using the alkaline extraction method can break the glycoprotein bond, which is based on the principle that the covalent bond between sugar and protein is unstable under alkaline conditions and is prone to breakage. However, the alkaline solution easily destroys the spatial structure and biological activity of polysaccharides, and the glycosamine bond in bird's nest polysaccharides is extremely easily destroyed by alkali.

[0060] In addition, specific enzymes can be used to break O-glycosidic bonds and N-glycosidic bonds to obtain polysaccharide components with higher purity. However, due to the extremely high cost of this specific enzyme, it is still only applied in the analysis field at present and is difficult to achieve large-scale production.

[0061] Based on the composition and structure of bird's nest, it is difficult to extract polysaccharides from bird's nest, and currently, there is no efficient and green extraction method for extracting bird's nest polysaccharides.

[0062] In some embodiments, a method for extracting bird's nest polysaccharides is provided, including the following steps:

[0063] Mix bird's nest with water, perform ultra-high pressure treatment, disperse, and prepare bird's nest homogenate;

[0064] Perform pulsed electric field treatment on the bird's nest homogenate, perform solid-liquid separation, and collect the supernatant; and,

[0065] Filter the supernatant, collect the filtrate and dry it to prepare bird's nest polysaccharides.

[0066] In some embodiments, a method for extracting bird's nest polysaccharides is provided, including the following steps:

[0067] S100: Mix bird's nest with water, perform ultra-high pressure treatment, disperse, and prepare bird's nest homogenate;

[0068] S200: Subject the bird's nest homogenate to pulsed electric field treatment, perform solid-liquid separation, and collect the supernatant; and,

[0069] S300: Filter the supernatant, collect the filtrate and dry it to prepare bird's nest polysaccharide.

[0070] In some embodiments, in step S100, purified water is added to the dried bird's nest such that the ratio of bird's nest to water is 1:20 to 1:70.

[0071] The component content inside the bird's nest is fixed, and the bird's nest has extremely strong water absorption. When the quality of the bird's nest remains unchanged, too low a ratio of purified water added will cause the bird's nest to not fully swell during subsequent processing, and the concentration difference inside and outside the bird's nest is small, resulting in a small mass transfer driving force and making it difficult to extract the active ingredients in the bird's nest. Conversely, too high a ratio of purified water also cannot further dissolve the active ingredients in the bird's nest.

[0072] In some embodiments, in step S100, the pressure of the ultra-high pressure treatment is 200 MPa to 500 MPa.

[0073] In some embodiments, in step S100, the time of the ultra-high pressure treatment is 5 to 15 min.

[0074] Ultra-high pressure can promote the penetration of purified water, cause the bird's nest to fully swell, and can also destroy the protein structure of the bird's nest, cause the protein to depolymerize, and help release the polysaccharide. However, too high a pressure will cause the release of other impurities and reduce the purity of the bird's nest polysaccharide.

[0075] In some embodiments, in step S100, during the dispersion step, the rotation speed of the dispersion is 8000 rpm to 12000 rpm.

[0076] In some embodiments, in step S100, during the dispersion step, the dispersion time is 1 min to 3 min.

[0077] Use a high-speed disperser to disperse the swollen bird's nest so that the bird's nest is broken up and dispersed into fine particle homogenate. High-speed dispersion is a strong mechanical force that can quickly destroy the structure of the bird's nest, break the bird's nest into fine particles, and help improve the treatment effect of pulsed electric field.

[0078] In some embodiments, in step S200, the electric field strength of the pulsed electric field treatment is 10 kV to 30 kV.

[0079] In some embodiments, in step S200, the number of pulses of the pulsed electric field treatment is 3 to 10.

[0080] In some embodiments, in step S200, the pulse width of the pulsed electric field treatment is 10 μs to 50 μs.

[0081] Research has shown that when the electric field strength exceeds 30 kV, electric sparks will be generated, which is not conducive to safe production. In addition, too high an electric field strength or too many pulse times will degrade polysaccharides and proteins, resulting in a decrease in the purity of bird's nest polysaccharides. The bonds connecting polysaccharides and proteins in bird's nest are mostly N-glycosidic bonds and O-glycosidic bonds. The bond energies of these two glycosidic bonds are less than that of peptide bonds, and the number of peptide bonds in bird's nest is much larger than that of N-glycosidic bonds and O-glycosidic bonds. Therefore, when a physical field of appropriate intensity is applied to bird's nest, the N-glycosidic bonds and O-glycosidic bonds break first, and the content of free polysaccharides in bird's nest increases, which helps to extract bird's nest polysaccharides. Moreover, due to the too large molecular weight of the proteins in bird's nest, after some peptide bonds are broken, compared with bird's nest polysaccharides, they are still substances with relatively large molecular weights and can be separated according to different molecular weights subsequently. In addition, pulsed electric fields can also destroy the gel structure of bird's nest, causing large molecular proteins to precipitate rapidly, which helps with subsequent separation.

[0082] In some embodiments, in step S200, the conditions for solid-liquid separation are centrifugation at 8000 - 10000 rpm for 20 - 30 min.

[0083] In some embodiments, step S300 includes the following steps:

[0084] S310: Microfilter the supernatant and collect the first filtrate;

[0085] S320: Ultrafilter the first filtrate.

[0086] In some embodiments, in step S310, the pore size of the microfiltration membrane is 4 μm - 6 μm.

[0087] In some embodiments, in step S310, the microfiltration membrane is a PVDF membrane.

[0088] The obtained supernatant is microfiltered to remove large molecular proteins in the supernatant. Since bird's nest is a gel-like substance, directly ultrafiltering the homogenized supernatant will form a gel-like thin film on the ultrafiltration membrane, which is not conducive to separation. Therefore, according to the characteristics of bird's nest, a microfiltration membrane with a pore size larger than that of the ultrafiltration membrane is first used for preliminary filtration and separation. The PVDF membrane has the characteristic of low adsorption, which can reduce the loss of substances in the solution.

[0089] In some embodiments, in step S320, during the ultrafiltration process, the cut-off molecular weight is 40 kDa - 50 kDa.

[0090] In some embodiments, in step S320, the centrifugal speed for ultrafiltration is 4000 rpm - 6000 rpm.

[0091] In some embodiments, in step S320, the ultrafiltration time is 20 min to 30 min.

[0092] In some embodiments, in step S320, the first filtrate is ultrafiltered in an ultrafiltration tube with a molecular weight cut-off (MWCO) of 40 kDa to 50 kDa.

[0093] Since the molecular weights of the proteins in the bird's nest after homogenization are mostly greater than 50 kDa, using a 50 kDa ultrafiltration membrane can separate the proteins and polysaccharides in the bird's nest to the greatest extent while reducing the loss of polysaccharides. In addition, the rotation speed is selected based on the centrifugal force tolerable by the ultrafiltration membrane.

[0094] In some embodiments, in step S300, the obtained filtrate is freeze-dried to obtain bird's nest polysaccharide.

[0095] The provided method for extracting bird's nest polysaccharide uses three high-energy physical fields, namely ultra-high pressure treatment, high-speed dispersion, and pulsed electric field, to pretreat the bird's nest to release the polysaccharides in the bird's nest to the greatest extent, and then obtains high-purity bird's nest polysaccharide through ultrafiltration. It can not only break the glycosidic bond to obtain free polysaccharides from glycoproteins, but also protect the structure and activity of polysaccharides. Compared with the current extraction methods, it eliminates the enzymatic biological method and the chemical methods such as ammonium sulfate precipitation and alcohol precipitation, saves the operation steps, greatly shortens the preparation time, and does not add any other substances to treat the bird's nest except purified water, avoiding the potential hazards of using organic solvents. In addition, the entire treatment process of this technology is at room temperature, avoiding the Maillard reaction of polysaccharides and proteins in the bird's nest during high-temperature treatment, thereby increasing the extraction difficulty. The extracted bird's nest polysaccharide has a high purity, up to 95.8%, which is much higher than the purity of the existing extracted bird's nest polysaccharide. The technical process of this invention is simple and easy to operate, providing new ideas and methods for the industrial production of bird's nest polysaccharide and its related products in the fields of food, medicine, skin care products, etc.

[0096] In some embodiments, a bird's nest polysaccharide is provided, and the monosaccharide composition includes the following components: N-acetylneuraminic acid, mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine.

[0097] In some embodiments, the monosaccharide composition of the edible bird's nest polysaccharide provided includes N-acetylneuraminic acid, mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Among them, the N-acetylneuraminic acid accounts for 31% - 32% of the total mass of the edible bird's nest polysaccharide, the mannose accounts for 7% - 8% of the total mass of the edible bird's nest polysaccharide, the fucose accounts for 1% - 2% of the total mass of the edible bird's nest polysaccharide, the galactose accounts for 25% - 26% of the total mass of the edible bird's nest polysaccharide, the N-acetylglucosamine accounts for 18% - 19% of the total mass of the edible bird's nest polysaccharide, and the N-acetylgalactosamine accounts for 11% - 12% of the total mass of the edible bird's nest polysaccharide.

[0098] In some embodiments, the edible bird's nest polysaccharide provided is prepared by the aforementioned method.

[0099] In some embodiments, there is provided the use of the edible bird's nest polysaccharide prepared by the aforementioned method or the aforementioned edible bird's nest polysaccharide in at least one of the products for scavenging free radicals, inhibiting Enterobacterales, improving the intestinal flora, and repairing cells.

[0100] In some embodiments of the provided use, the products include one or more of foods, medicines, and skin care products.

[0101] The embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used

[0102] to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, priority is given to the guidance given in the present invention, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0103] In the following specific examples, for the measurement parameters of the raw material components, there may be slight deviations within the weighing accuracy range without special instructions. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0104] The sources and models of the instruments used are as follows: Automatic Kjeldahl nitrogen analyzer, FOSS, Denmark, KJELTEC 8400; Ultraviolet-visible spectrophotometer, Jena, Germany, SPECORD® 50 PLUS; Liquid chromatograph, Shimadzu, Japan, LC-20AD; Gas chromatography-mass spectrometry, GCMS-TQ8040, Shimadzu, Japan; Nitrogen evaporator, CM-12, Beijing Chengmeng Weiye Technology Co., Ltd.

[0105] Example 1

[0106] Purified water is added to dry bird's nest to make the ratio of bird's nest to water 1:30. It is subjected to ultra-high pressure treatment at a pressure of 400 MPa for a holding time of 10 min. The ultra-high pressure-treated bird's nest is dispersed at 10,000 rpm for 2 min using a high-speed disperser to obtain bird's nest homogenate; then, the bird's nest homogenate is sent to the pulsed electric field treatment chamber through a peristaltic pump, with the electric field strength set at 20 kV, the pulse width at 40 μs, and the number of pulses at 8. After pulsed electric field treatment, the bird's nest homogenate is centrifuged at 10,000 rpm for 25 min, the supernatant is filtered through a 5-μm PVDF membrane, and the filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa for 25 min at a rotation speed of 4,000 rpm. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0107] The composition of the obtained bird's nest polysaccharide is determined. The crude polysaccharide content in the sample is determined by the phenol-sulfuric acid method, the sialic acid content in the sample is determined by GB 31614.1-2023 "National Food Safety Standard - Determination of Sialic Acid in Foods", the protein content in the sample is determined by GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods", and the ash content in the sample is determined by GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Foods". The above results are all calculated on a dry basis. The polysaccharide content in the sample includes the crude polysaccharide content and sialic acid content determined by the above methods. The purity is expressed as the percentage of the polysaccharide mass in the total mass. The results are shown in Table 1.

[0108] Table 1

[0109]

[0110] As can be seen from the results in Table 1, the total content of crude polysaccharide and sialic acid in the extracted bird's nest polysaccharide accounts for 95.8%, and the purity is relatively high.

[0111] Example 2

[0112] Purified water is added to dry bird's nest to make the ratio of bird's nest to water 1:30. It is subjected to ultra-high pressure treatment at a pressure of 250 MPa for a holding time of 8 min. The ultra-high pressure-treated bird's nest is dispersed at 10,000 rpm for 2 min using a high-speed disperser to obtain bird's nest homogenate; then, the bird's nest homogenate is sent to the pulsed electric field treatment chamber through a peristaltic pump, with the electric field strength set at 20 kV, the pulse width at 40 μs, and the number of pulses at 8. After pulsed electric field treatment, the bird's nest homogenate is centrifuged at 10,000 rpm for 25 min, the supernatant is filtered through a 5-μm PVDF membrane, and the filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa for 25 min at a rotation speed of 4,000 rpm. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0113] Example 3

[0114] Purified water is added to dried bird's nest so that the ratio of bird's nest to water is 1:30. It is subjected to ultra-high pressure treatment at a pressure of 400 MPa for a holding time of 10 min. The bird's nest after ultra-high pressure treatment is dispersed by a high-speed disperser at 10,000 rpm for 2 min to obtain a bird's nest homogenate; then, the bird's nest homogenate is sent to a pulsed electric field treatment chamber through a peristaltic pump, and the electric field strength is set at 10 kV, the pulse width is 40 μs, and the number of pulses is 5. After pulsed electric field treatment, the bird's nest homogenate is centrifuged at 10,000 rpm for 25 min, and the supernatant is filtered through a 5-μm PVDF membrane. The filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa at a rotation speed of 4,000 rpm for 25 min. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0115] Comparative Example 1

[0116] Purified water is added to dried bird's nest so that the ratio of bird's nest to water is 1:30. It is subjected to ultra-high pressure treatment at a pressure of 400 MPa for a holding time of 10 min. The treated bird's nest is centrifuged at 10,000 rpm for 25 min, and the supernatant is filtered through a 5-μm PVDF membrane. The filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa at a rotation speed of 4,000 rpm for 25 min. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0117] Comparative Example 2

[0118] Purified water is added to dried bird's nest so that the ratio of bird's nest to water is 1:30. The bird's nest is sent to a pulsed electric field treatment chamber through a peristaltic pump, and the electric field strength is set at 20 kV and the number of pulses is 8. After pulsed electric field treatment, the bird's nest is centrifuged at 10,000 rpm for 25 min, and the supernatant is filtered through a 5-μm PVDF membrane. The filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa at a rotation speed of 4,000 rpm for 25 min. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0119] Comparative Example 3

[0120] Purified water is added to dried bird's nest so that the ratio of bird's nest to water is 1:30. It is subjected to ultra-high pressure treatment at a pressure of 400 MPa for a holding time of 10 min. Then, the treated one is sent to a pulsed electric field treatment chamber through a peristaltic pump, and the electric field strength is set at 20 kV and the number of pulses is 8. After pulsed electric field treatment, the bird's nest is centrifuged at 10,000 rpm for 25 min, and the supernatant is filtered through a 5-μm PVDF membrane. The filtered liquid is ultrafiltered through an ultrafiltration tube with MWCO = 50 kDa at a rotation speed of 4,000 rpm for 25 min. The lower-layer liquid after ultrafiltration is collected and freeze-dried to obtain bird's nest polysaccharide.

[0121] Experimental Example 1

[0122] Determine the polysaccharide contents in Examples 1 to 3 and Comparative Examples 1 to 3. The crude polysaccharide content in the samples was determined by the phenol-sulfuric acid method, and the sialic acid content in the samples was determined by GB 31614.1-2023 "National Food Safety Standard - Determination of Sialic Acid in Foods". The polysaccharide content in the samples includes the crude polysaccharide content and the sialic acid content determined by the above methods. The purity is expressed as the percentage of the polysaccharide mass to the total mass. The results are shown in Table 2.

[0123] Table 2 Purity of the bird's nest polysaccharides extracted under different conditions

[0124]

[0125] From the results in Table 2, it can be seen that Example 1 has the highest purity of bird's nest polysaccharides, which is 95.8%. When the treatment intensity of ultra-high pressure decreases, the purity of bird's nest polysaccharides also decreases to 93.3%. When the treatment intensity of pulsed electric field decreases, the purity of bird's nest polysaccharides decreases to 91.0%. In addition, the purities of the bird's nest polysaccharides extracted by single physical field or the combination of two physical fields are further reduced by 82.7%, 84.5% and 88.6% respectively, indicating that high-speed dispersion, ultra-high pressure and pulsed electric field treatment play an indispensable and important role in improving the purity of bird's nest polysaccharides.

[0126] Conclusion: Purity is one of the important indicators for evaluating the quality of extract products. In the examples, three physical fields were combined for treatment, and in the comparative examples, one or two of the physical fields were used for treatment. The results show that compared with single physical field or the combination of two physical fields, the purity of the bird's nest polysaccharides extracted by the combination of three physical fields is higher. The combination of three physical fields can maximize the purity of the bird's nest polysaccharides, and each physical field plays an important role. Ultra-high pressure can promote the penetration of purified water, fully swell the bird's nest, and can also destroy the protein structure of the bird's nest, depolymerize the protein, and contribute to the release of polysaccharides. High-speed dispersion can quickly destroy the structure of the bird's nest, break the bird's nest into fine particles, and contribute to improving the treatment effect of pulsed electric field. Finally, combined with pulsed electric field treatment, the glycosidic bonds connecting polysaccharides and proteins are broken to extract high-purity polysaccharides.

[0127] Test Example 2

[0128] The monosaccharide composition of the polysaccharide obtained in Example 1 was further analyzed. Specifically, 10.0 mg of the sample powder that had passed through a 100-mesh sieve was accurately weighed and placed in a 10 mL hydrolysis tube. 2 mL of 1.0 mol / L trifluoroacetic acid solution and 0.10 mL of 1 mg / mL inositol internal standard were added. After filling with nitrogen, it was sealed and subjected to acid hydrolysis at 110 °C in an electrothermal constant temperature drying oven for 4 h. After the hydrolysis solution cooled to room temperature, it was transferred to a nitrogen blowing tube, and trifluoroacetic acid was completely removed by nitrogen blowing at 80 °C, and then redissolved with 1 mL of water. 100 μL of the hydrolyzed sample was taken and added to 100 μL of 20 mg / mL hydroxylamine hydrochloride pyridine solution, and the tube was sealed and reacted at 90 °C for 30 min. After cooling to room temperature, 1 mL of acetic anhydride was added, and the tube was sealed and reacted at 90 °C for 30 min. After cooling, it was nitrogen blown dry at 80 °C, and 1.00 mL of chloroform was added to dissolve it, and it was diluted 100 times and then subjected to gas chromatography-mass spectrometry analysis. A Shimadzu quartz capillary chromatographic column (SH-Rtx-1701, 30 m × 0.25 mm, 0.25 μm) was used. The initial temperature was 160 °C (2 min), and it was heated to 230 °C (20 min) at a heating rate of 11 °C / min, and then heated to 250 °C (5 min) at a heating rate of 10 °C / min. The injection temperature was 240 °C, the interface temperature was 240 °C, the ion source temperature was 230 °C, the ionization energy was 70 eV, the ionization mode was EI, the injection volume was 1 μL, the flow rate was 1.5 mL / min, the injection mode was splitless, and the carrier gas was helium. The sialic acid content in the sample was determined according to GB 31614.1-2023 "National Food Safety Standard - Determination of Sialic Acid in Foods". The results are as Figure 1 , Figure 2 and Table 3 show, among which, Figure 1 is the gas chromatogram of the monosaccharide composition of the bird's nest polysaccharide obtained in Example 1, Figure 2 is the liquid chromatogram of N-acetylneuraminic acid in the obtained bird's nest polysaccharide, and Table 3 shows the monosaccharide composition of the bird's nest polysaccharide.

[0129] Table 3 Monosaccharide Composition of Bird's Nest Polysaccharide

[0130]

[0131] As Figure 1 , Figure 2 and Table 3 show, the monosaccharide composition of the bird's nest polysaccharide includes N-acetylneuraminic acid, mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Among them, N-acetylneuraminic acid accounts for 31.6% of the total mass, mannose accounts for 7.1% of the total mass, fucose accounts for 1.8% of the total mass, galactose accounts for 25.4% of the total mass, N-acetylglucose accounts for 18.6% of the total mass, and N-acetylgalactosamine accounts for 11.3% of the total mass.

[0132] Test Example 3

[0133] 1. Detection method

[0134] The DPPH free radical scavenging ability, antibacterial ability, and cell scratch repair ability of the bird's nest polysaccharide obtained in Example 1 were determined by the following methods. The specific operations are as follows:

[0135] (1)Evaluation of DPPH free radical scavenging ability

[0136] Preparation of DPPH free radical solution: Prepare a 0.04 mg / mL DPPH solution by dissolving DPPH in absolute ethanol. Sample preparation: Prepare different concentration solutions (50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2500 μg / mL) of the crude bird's nest polysaccharide sample using ultrapure water.

[0137] Preparation of the solution to be measured: A0) Add 2 mL of absolute ethanol and 2 mL of DPPH free radical solution to a test tube, A1) Add 2 mL of the sample solution and 2 mL of DPPH free radical solution to a test tube, A2) Add 2 mL of the sample solution and 2 mL of absolute ethanol to a test tube. Mix the above solutions to be measured evenly and let them stand at room temperature for 30 min. Finally, centrifuge the mixed solution at 5000 r / min for 10 min. Measure the absorbance value of the supernatant of the centrifuged solution at 517 nm to calculate the scavenging rate of the sample for DPPH free radicals. The scavenging rate of the sample for DPPH free radicals is calculated using the following formula:

[0138] ;

[0139] where A0: the absorbance value of 2 mL of absolute ethanol + 2 mL of DPPH free radical solution;

[0140] A1: the absorbance value of 2 mL of the sample solution + 2 mL of DPPH free radical solution;

[0141] A2: the absorbance value of 2 mL of the sample solution + 2 mL of absolute ethanol.

[0142] (2)Evaluation of cell scratch repair

[0143] Seed HaCaT cells in the logarithmic growth phase on a 6-well plate, 6×10 per well 5Seed the cells at a density of [number] and place them in an incubator at 37 °C with 5% CO₂ for 24 h to allow the cells to adhere completely. Subsequently, use a pipette tip to create a cell scratch in the cell culture plate. Remove the culture medium and wash twice with PBS buffer. Add fresh medium to the normal control wells and fresh medium containing the edible bird's nest polysaccharide sample obtained in Example 1 at the corresponding concentration to the sample wells, and take a photo. Then place it in an incubator at 37 °C with 5% CO₂ and take photos regularly (12 h, 24 h). Use Image J to analyze the scratch area of each group and calculate the healing area (Y). The calculation formula is as follows:

[0144] .

[0145] 2. Test results

[0146] (1)Evaluation of DPPH free radical scavenging ability

[0147] The scavenging rates of the edible bird's nest polysaccharide extracted in Example 1 on DPPH free radicals are shown in Table 4 below and Figure 3 as follows.

[0148] Table 4

[0149]

[0150] As can be seen from Table 4 and Figure 3 when the concentration of the edible bird's nest polysaccharide is 0.2 - 1.0 mg / mL, the scavenging rate of DPPH free radicals is 60.9% - 89.6%. As the concentration of the edible bird's nest polysaccharide increases, the scavenging rate of DPPH free radicals increases. When the concentration of the edible bird's nest polysaccharide is 1.0 mg / mL, the scavenging rate of DPPH free radicals reaches 89.6%, indicating that the edible bird's nest polysaccharide extracted in the present invention has good antioxidant ability and has the potential to be applied in functional foods, skin care products, and pharmaceuticals.

[0151] (2)Evaluation of cell scratch repair

[0152] The test results of the repair effect of the edible bird's nest polysaccharide on HaCaT cells are shown in Table 5 and Figure 4 as follows.

[0153] Table 5 Repair effect of the edible bird's nest polysaccharide on HaCaT cells

[0154]

[0155] As can be seen from Table 5 and Figure 4It can be seen that when the concentration of the edible bird's nest polysaccharide is 0.5 - 2.5 mg / mL, the repair rate for HaCaT cells is 30.7% - 76.7%. As the concentration of the edible bird's nest polysaccharide increases, the repair rate for HaCaT cells increases. When the concentration of the edible bird's nest polysaccharide is 2.5 mg / mL, the repair rate reaches 76.7%, indicating that the edible bird's nest polysaccharide extracted in the present invention has good repair ability and has the potential to be applied in functional foods, skin care products, and pharmaceuticals.

[0156] Conclusion: The above DPPH free radical scavenging experiment and cell repair experiment prove that the edible bird's nest polysaccharide has good DPPH free radical scavenging ability and cell repair ability, and has multiple physiological activities.

[0157] Test Example 4

[0158] The following method was used to conduct an in vitro simulated fermentation experiment on the edible bird's nest polysaccharide obtained in Example 1, and the fermentation broth was collected for sequencing analysis. The specific operations are as follows:

[0159] (1) Collection of fecal samples

[0160] Volunteers providing fecal samples need to meet the following conditions: body mass index BMI is within the normal range (18.5 < BMI < 25); no digestive system diseases, dietary restrictions, endocrine diseases, inflammatory diseases, and other chronic diseases, etc.; within the past 3 months, a regular diet, regular bowel movements, and no physical discomfort; no antibiotics were taken within 3 months before sampling, and no prebiotic products were taken within 1 month.

[0161] (2) Preparation of fecal suspension

[0162] Pick an adequate amount of fresh fecal samples, dispense them into sampling tubes and tighten the lids, quickly transfer them to an anaerobic incubator, and then add PBS medium (pH 7.3) that has been autoclaved at 121 °C. Mix the fecal suspension thoroughly and adjust its concentration to 25% (w / v). After filtering through four layers of gauze, it is reserved for use.

[0163] (3) In vitro simulated fermentation

[0164] In a 250 mL fermentation flask, 50 mg of the sample, 4.8 mL of BCM culture medium (2.0 g / L yeast extract, 3.0 g / L tryptone, 2.0 g / L peptone, 0.5 g / L bile salt No. 3, 0.5 g / L L-H-Cys-OH HCl, 0.05 g / L erythromycin chloride, 0.1 g / L NaCl, 0.4 g / L MgCl2·6H2O, 0.01 g / L CaCl2·6H2O, 0.4 g / L KH2PO4, 1 mL / L Tween 80, and 2 mL / L trace element solution were added in sequence. The pH was adjusted to 7.3. It was sterilized at 121 °C for 20 min.), and 0.2 mL of fecal suspension were added. After thorough mixing, the flask was sealed with a lid and transferred to a 37 °C air bath shaker incubator for 48 h.

[0165] (4) Analysis of fermentation products

[0166] DNA was extracted from the fermentation products for 16S rRNA analysis.

[0167] PCoA analysis was performed on the intestinal flora of the blank group and the bird's nest polysaccharide group, and the results are as Figure 5 and Figure 6 shown. Figure 5 The results shown indicate that the addition of bird's nest polysaccharide caused obvious changes in the intestinal flora, and there were obvious differences between the flora. Further analysis of the intestinal flora showed that, as can be seen from Figure 6 , compared with the blank group, the abundance of Bacteroidaceae increased and the abundance of Enterobacteriaceae decreased in the intestinal flora composition after fermentation with bird's nest polysaccharide. Previous studies have found that Bacteroides have a symbiotic relationship with humans. They help break down food and produce the nutrients and energy required by the body. Salmonella, Shigella, Escherichia, etc. in Enterobacteriaceae are widely pathogenic to the host and can cause intestinal disorders or intestinal inflammation. The increase in their abundance leads to intestinal ecological imbalance. The above results indicate that bird's nest polysaccharide can improve the host's intestinal flora and maintain intestinal health by increasing the abundance of beneficial bacteria and decreasing the abundance of harmful bacteria.

[0168] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0169] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for extracting bird's nest polysaccharide, characterized in that, It includes the following steps: Mix the bird's nest with water, perform ultra-high pressure treatment, disperse, and prepare bird's nest homogenate; Perform pulsed electric field treatment on the bird's nest homogenate, carry out solid-liquid separation, and collect the supernatant; And, Filter the supernatant, collect the filtrate and dry it to prepare bird's nest polysaccharide.

2. The method for extracting bird's nest polysaccharide according to claim 1, wherein Meet one or more of the following conditions (1) to (5): (1) The mass ratio of the bird's nest to the water is 1:(20 - 70); (2) The pressure of ultra-high pressure treatment is 200 MPa - 500 MPa; (3) The time of ultra-high pressure treatment is 5 min - 15 min; (4) The rotation speed of dispersion is 8000 rpm - 12000 rpm; and, (5) The time of dispersion is 1 min - 3 min.

3. The method for extracting edible bird's nest polysaccharide according to claim 1 or 2, characterized in that Meet one or more of the following conditions (1) to (4): (1) The electric field strength of pulsed electric field treatment is 10 kV - 30 kV; (2) The number of pulses of pulsed electric field treatment is 3 - 10; (3) The pulse width of pulsed electric field treatment is 10 μs - 50 μs; and, (4) The condition of solid-liquid separation is centrifugation at 8000 rpm - 10000 rpm for 20 min - 30 min.

4. The method for extracting bird's nest polysaccharide according to any one of claims 1 to 3, characterized in that, Performing the filtration includes microfiltering the supernatant, collecting the first filtrate, and ultrafiltering the first filtrate.

5. The method for extracting bird's nest polysaccharide according to claim 4, characterized in that, The pore size of the microfiltration membrane is 4 μm - 6 μm.

6. The method for extracting bird's nest polysaccharide according to claim 4, wherein, Meet one or more of the following conditions (1) to (3): (1) During ultrafiltration, the molecular weight cut-off is 40 kDa - 50 kDa; (2) The centrifugation speed of ultrafiltration is 4000 rpm - 6000 rpm; and, (3) The time of ultrafiltration is 20 min - 30 min.

7. A bird's nest polysaccharide, characterized in that, The monosaccharide composition of the bird's nest polysaccharide includes the following components: The monosaccharide composition of the bird's nest polysaccharide includes N-acetylneuraminic acid, mannose, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Among them, the N-acetylneuraminic acid accounts for 31% - 32% of the total mass of the bird's nest polysaccharide, the mannose accounts for 7% - 8% of the total mass of the bird's nest polysaccharide, the fucose accounts for 1% - 2% of the total mass of the bird's nest polysaccharide, the galactose accounts for 25% - 26% of the total mass of the bird's nest polysaccharide, the N-acetylglucosamine accounts for 18% - 19% of the total mass of the bird's nest polysaccharide, and the N-acetylgalactosamine accounts for 11% - 12% of the total mass of the bird's nest polysaccharide.

8. The edible bird's nest polysaccharide according to claim 7, characterized in that, The bird's nest polysaccharide is prepared by the method according to any one of claims 1 - 6.

9. Use of the bird's nest polysaccharide prepared by the method according to any one of claims 1 - 6 or the bird's nest polysaccharide according to claim 7 or 8 in the preparation of at least one of the products for scavenging free radicals, improving intestinal flora, and repairing cells.

10. The application according to claim 9, characterized in that, The product includes one or more of food, medicine, and skin care products.

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