Method for preparing sialic acid oligosaccharide-containing composition

Abstract

The present invention relates to a method for preparing a sialyl oligosaccharide-containing composition. The method comprises the following steps: grinding cubilose, and then adding lactose; the sialic acid oligosaccharide-containing composition is characterized in that sialic acid oligosaccharide is taken as a raw material, the sialic acid oligosaccharide is added into the sialic acid oligosaccharide-containing composition, the sialic acid oligosaccharide-containing composition is added into the sialic acid oligosaccharide-containing composition, the sialic acid oligosaccharide-containing composition is added into the sialic acid oligosaccharide-containing composition, the sialic acid oligosaccharide-containing composition is heated and stirred, the sialic acid oligosaccharide-containing composition is fully reacted, the amino acid sequence of the LhSiase is shown as SEQ ID NO.1, the final concentration of the LhSiase is 0.1 U / mL to 1 U / mL, the final concentration of the lactose is 1 w / v% to 10 w / v%, and the final concentration of the crushed cubilose is 0.25 w / v% to 2.5 w / v%. According to the method, the bioavailability of the cubilose is improved, and the reutilization of wastes is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of enzyme processing, in particular to a method for preparing a sialyl oligosaccharide-containing composition. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk, which can be divided into fucosylated, sialylated and non-fucosylated oligosaccharides. They have important physiological functions, can promote the growth of beneficial bacteria in the infant's intestine, enhance the gastrointestinal barrier, prevent infection, and provide potential support for the immune system, brain and cognitive development. Sialyl oligosaccharides are an important component of HMOs, mainly in the form of 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), which have many health benefits for infants, including promoting brain development, improving cognition and preventing infection. However, HMOs are almost non-existent in cow's milk (or any animal milk).

[0003] Currently, the synthesis methods of sialyl oligosaccharides mainly include chemical method and enzymatic method. The chemical method usually involves complex protection and deprotection steps, and sialic acid is a nine-carbon acidic sugar, which is more difficult to modify than common six-carbon sugars. In the enzymatic synthesis, sialyltransferase can be used to synthesize sialyl oligosaccharides, but due to the need for expensive glycosyl donors and strict substrate selectivity, its large-scale application is limited.

[0004] Edible bird's nest, also known as edible bird's nest, edible bird's nest, and edible bird's nest, is produced by the swiftlet, which mainly inhabits Southeast Asian countries (Indonesia, Malaysia, Philippines, Thailand, etc.). Edible bird's nest is known as "the Eastern caviar". "Benjing Fengyuan" says: "Edible bird's nest, sweet and non-toxic, can make gold and water coexist, kidney qi can be added to the lungs, and stomach qi can also be stable, now people use it to adjust and supplement the labor, cough and red sputum". Studies have shown that the main chemical components of edible bird's nest are: water, lipids, sialic acid (considered the most critical active ingredient in edible bird's nest), proteins, carbohydrates (sugars and proteins exist in the form of glycoproteins), amino acids, fiber, inorganic substances, etc. Edible bird's nest will produce a large amount of edible bird's nest during processing, storage and transportation. This kind of debris is difficult to integrate into a bowl, has low commodity value, and leads to low actual utilization rate.

[0005] Currently, edible bird's nest is cooked by traditional method, i.e., soaking and stewing. However, the preparation is cumbersome and inconvenient, and the types of bird's nest products on the market are single, which cannot meet the needs of modern urban fast-paced life and nutritional health. Moreover, most of the sialic acid in bird's nest is sialic acid glycoprotein, and the molecular weight is basically greater than 100 kDa. Studies have shown that the absorption rate of human body to directly edible bird's nest is only about 20%, which is closely related to the special glycoprotein structure in bird's nest. The sugar chains on the surface of glycoprotein form steric hindrance, which hinders the recognition and decomposition of digestive enzymes to protein polypeptide chain, thereby significantly reducing the absorption efficiency of human body to bird's nest protein. After in vitro simulated static digestion, 55% of sialic acid in water bath stewed bird's nest remains in the undissolved gel solid, which shows that the utilization rate of sialic acid in bird's nest still needs to be improved.

[0006] Therefore, there is an urgent need for an efficient, convenient and economical processing method which can improve the utilization rate of bird's nest and the absorption rate of human body to bird's nest. SUMMARY

[0007] Therefore, the main purpose of the present application is to improve the product value of bird's nest fragments and convert the high-content sialic acid glycoprotein in bird's nest fragments into sialic acid oligosaccharide which is more beneficial to human body absorption.

[0008] The specific technical solutions of the present application are as follows.

[0009] The present application provides a method for preparing a sialic acid oligosaccharide-containing composition, comprising the following steps:

[0010] Grinding the bird's nest fragments, then adding lactose; adding Lh Siase1 for reaction, and obtaining the sialic acid oligosaccharide-containing composition after the reaction is completed, wherein, Lh The amino acid sequence of Siase1 is shown in SEQ ID NO. 1; Lh The final concentration of Siase1 is 0.1 U / mL to 1 U / mL, the final concentration of lactose is 1 w / v% to 10 w / v%, and the final concentration of bird's nest fragments is 0.25 w / v% to 2.5 w / v%.

[0011] In some embodiments, Lh The final concentration of Siase1 is 0.3 U / mL to 0.5 U / mL.

[0012] In some embodiments, the final concentration of lactose is 4 w / v% to 7 w / v%.

[0013] In some embodiments, the final concentration of bird's nest fragments is 0.5 w / v% to 1.5 w / v%.

[0014] In some embodiments, the temperature of the reaction is 35°C to 45°C, and the time of the reaction is 0.5 h to 24 h.

[0015] In some implementations, the reaction time with heating and stirring is 1 to 5 hours.

[0016] In some implementations, bird's nest fragments are derived from the residue after bird's nest processing.

[0017] The present invention also provides an amino acid sequence as shown in SEQ ID NO.1. Lh The use of Siase1 in the preparation of bird's nest powder preparations containing sialic acid oligosaccharides, wherein the bird's nest powder preparations are prepared by the following method: grinding bird's nest powder, then adding lactose; adding... Lh Siase1 undergoes a reaction, and after the reaction is complete, a bird's nest powder preparation is obtained. Lh The final concentration of Siase1 was 0.1 U / mL to 1 U / mL.

[0018] The beneficial effects of this invention are:

[0019] On the one hand, in this invention, the source is Lactobacillus helsingborgensis Specific in WP_109917843.1 Lh Siase1, when processed with bird's nest fragments and combined with a certain concentration of lactose, can not only synthesize sialic acid oligosaccharides in one step, but also has a significantly better processing effect on bird's nest fragments than the same enzyme.

[0020] On the other hand, the present invention uses further ground bird's nest fragments as raw materials to convert the insoluble sialic acid glycoprotein in the bird's nest fragments into small molecule sialic acid oligosaccharides, thereby enhancing human absorption and improving the bioavailability of bird's nest fragments.

[0021] On the other hand, the sialic acid oligosaccharides synthesized by this invention are purer and produce fewer byproducts, which greatly improves their utilization rate in the human body.

[0022] On the other hand, this invention uses bird's nest fragments, which have lower commercial value, as raw materials, making it more economical and faster than existing methods and realizing the reuse of waste. Attached Figure Description

[0023] Figure 1 For the sialidase of the present invention ( Lh SDS-PAGE image of Siase1, where: M represents the standard molecular weight of the protein; 1 represents... Lh The crude enzyme solution of the recombinant bacteria Siase1; 2 is the pure enzyme solution obtained by nickel ion affinity chromatography.

[0024] Figure 2 For the measurements under different temperature conditions in Example 2 Lh The result of Siase1 enzyme activity.

[0025] Figure 3 Results of Siase1 enzyme activity for Example 2 under different pH conditions Lh Results of Siase1 enzyme activity for Example 2 under different pH conditions

[0026] Figure 4 Results of Siase1 enzyme activity for Example 2 under different metal ions Lh Results of Siase1 enzyme activity for Example 2 under different metal ions

[0027] Figure 5 Liquid chromatography of sialylated oligosaccharides produced by the method of the present application compared to sialylated oligosaccharide standards.

[0028] Figure 6 Results of sialylated oligosaccharides produced under different reaction conditions for Example 4. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described in detail below with reference to the specific embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] Unless otherwise defined, 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 application belongs. The following definitions supplement those in the art and are intended to apply only to this application and are not intended to limit the scope or practice of any related or related art, e.g., a commonly used patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice testing, the materials and methods described herein are preferred. Therefore, the terms used herein are intended to describe only the specific embodiments and are not intended to limit the present application.

[0031] In the present application, the terms "comprise", "include", "have" are open-ended descriptions, containing the specified steps described, and other steps which do not materially affect, are optional and are not excluded.

[0032] In the present application, the term "comprise", "include", "have" are open-ended descriptions, containing the specified steps described, and other steps which do not materially affect, are optional and are not excluded. Lh Siase1, by one-step conversion catalysis to produce sialylated oligosaccharides, thus completing the present application.

[0033] The first aspect of the present application provides a method for preparing a sialylated oligosaccharide-containing composition, comprising the following steps:

[0034] The bird's nest is ground, then lactose is added; Siase1 is added LhSiase1 was reacted, and after the reaction was completed, a composition containing sialic acid oligosaccharides was obtained.

[0035] There is no significant difference in nutritional value between broken bird's nest and whole bird's nest, but the sialic acid content is particularly rich, reaching about 12% to 15%. In this invention, broken bird's nest is used as a raw material, which can provide sialic acid glycosyl donors while greatly saving production costs.

[0036] In some implementations, bird's nest fragments are derived from the residue after bird's nest processing.

[0037] In some implementations, the bird's nest fragments are ground into a fine powder of 50-100 mesh. For example, the ground bird's nest fragments can be 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, or 100 mesh.

[0038] Sialidase (EC. 3.2.1.18) is a glycoside hydrolase with diverse sources and varying functions. For example, sialidase on the surface of influenza viruses can cleave sialic acid on glycoproteins and glycolipids on the host cell surface; many pathogenic bacteria (such as Vibrio cholerae, Streptococcus pneumoniae, and Streptococcus stomatitis) also secrete sialidase to expose receptors on the host cell surface that are masked by sialic acid, facilitating bacterial adhesion. In the food industry, sialidase has been reported to be used to modify milk proteins and improve their functional properties, such as emulsifying and foaming properties.

[0039] In this invention, based on extensive preliminary research, [the following were selected]. Lactobacillus helsingborgensis (WP_109917843.1) Sialidase, and the sialidase gene ( Lh Siase1 was cloned and expressed in E. coli BL21(DE3) to obtain sialidase ( Lh Siase1).

[0040] Compared to sialidases from other sources, the sialidase selected in this invention ( Lh Siase1 not only efficiently hydrolyzes sialic acid glycoproteins in bird's nest fragments, but also exhibits significant transglycosylation activity, enabling efficient synthesis of sialic acid oligosaccharides. Furthermore, the sialysinase (Siase1) of this invention... Lh Siase1, when bird's nest fragments are used as the processing raw material, has a significantly higher content of synthetic sialic acid oligosaccharides than other types of sialic acid sidases.

[0041] In an embodiment of the present invention, Lh The amino acid sequence of Siase1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0042] SEQ ID NO.1:

[0043] MELQESLTAKFDRTMLLPDSGIQSHASNLLILPNGDTLCTWFSGTEEGTDDITIFMAELKAGTDSWSKPVPMSNNPGRSDQNPVLFAAPDGDIWLLYTSQVGGNEDTAIIQYRVSHDNGKTWSLQSNLFPGESGLFIRHPLVVTANGEWLLPLYHCVVKNDGKPWDGSYDYSCVRVSQDSGQTWKEYEVPDSQGCVQMSIVKLQNNAGYVGFFRSRWADNIYRSFSNDGSRWTPPKAIDLPNNNSSIQAALLPDGNIILAFNKSSAEDATVRRLSLFSEKTTAKETSNKKAAKTAFWGAPRAPLTVAISEDNGKTWPYVRNIAEGSGYALTNNSKDKKNREFSYPSVKPTRNGKIALTFTYFRQNIAFVEFTEDWVKEEKE

[0044] SEQ ID NO. 2:

[0045]

[0046] In some implementation schemes, Lh Siase1 was prepared by the following method:

[0047] will come from Lactobacillus helsingborgensis. Sialidase of WP_109917843.1 ( Lh The gene of Siase1 was introduced into plasmid pET28a(+), which was then introduced into competent Escherichia coli BL21(DE3) to obtain recombinant E. coli. The recombinant E. coli was then cultured to scale up, and after induction culture, the E. coli were collected by centrifugation, disrupted, and the supernatant was collected and purified to obtain... Lh Siase1.

[0048] In some implementations, the culture medium used for expansion culture is LB medium.

[0049] In some embodiments, the culture medium also includes kanamycin sulfate at a concentration of 40 µg / mL to 50 µg / mL.

[0050] In some implementations, induction culture is carried out after expansion culture by adding isopropyl thiogalactoside (IPTG) to the culture medium.

[0051] In some implementations, the final concentration of IPTG in the culture medium is 0.1 mM to 0.2 mM.

[0052] In some implementations, the induction culture conditions are 4°C to 8°C, 150 rpm to 200 rpm, and culture for 36 to 48 hours.

[0053] In some implementations, LhSiase1 is purified using a nickel ion affinity chromatography column.

[0054] In some implementation schemes, Lh The final concentration of Siase1 is from 0.1 U / mL to 1 U / mL. For example, the final concentration of LhSiase1 can be 0.1 U / mL, 0.2 U / mL, 0.3 U / mL, 0.6 U / mL, 0.7 U / mL, 0.8 U / mL, 0.9 U / mL or 1 U / mL.

[0055] In a preferred embodiment, Lh The final concentration of Siase1 was 0.3 U / mL to 0.5 U / mL.

[0056] In some embodiments, the final concentration of lactose is 1 w / v% to 10 w / v%. For example, the final concentration of lactose can be 1 w / v%, 2 w / v%, 3 w / v%, 4 w / v%, 5 w / v%, 6 w / v%, 7 w / v%, 8 w / v%, 9 w / v% or 10 w / v%.

[0057] In preferred embodiments, the final concentration of lactose is 4 w / v% to 7 w / v%.

[0058] In some embodiments, the final concentration of bird's nest pieces is 0.25 w / v% to 2.5 w / v%. For example, the final concentration of bird's nest pieces can be 0.25 w / v%, 0.3 w / v%, 0.5 w / v%, 1.5 w / v%, 2 w / v% or 2.5 w / v%.

[0059] By selecting the above specific ranges of addition of LhSiase 1, lactose, and bird's nest pieces, the yield of sialyl-oligosaccharides obtained can be improved, maximizing the benefits.

[0060] In a preferred embodiment, the final concentration of bird's nest pieces is 0.5 w / v% to 1.5 w / v%.

[0061] In some embodiments, the temperature of the reaction is 35°C to 45°C. For example, the temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or 45°C.

[0062] In some embodiments, the time of the reaction is 0.5 h to 24 h. For example, the time can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 5 h, 8 h, 10 h, 12 h, 15 h, 20 h or 24 h.

[0063] In preferred embodiments, the time of the reaction is 1 h to 5 h.

[0064] In some embodiments, the pH of the reaction is 4.0 to 7.0. For example, the pH can be 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0. In a particular embodiment, the pH of the reaction is 5.0.

[0065] In some embodiments, agitation is performed during the reaction.

[0066] In particular embodiments, the sialyl-oligosaccharide-containing composition can be used as a bird's nest pieces reconstituting agent.

[0067] The present application also provides a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 1 LhThe use of Siase1 in the preparation of bird's nest powder preparations containing sialic acid oligosaccharides, wherein the bird's nest powder preparations are prepared by the following method: grinding bird's nest powder, then adding lactose; adding... Lh Siase1 is heated and stirred to allow for a full reaction. After the reaction is complete, bird's nest powder is obtained as a ready-to-use preparation.

[0068] The above-described technical solution in the method for preparing compositions containing sialic acid oligosaccharides of the present invention is also applicable to... Lh Use of Siase1 in the preparation of bird's nest powder concentrate containing sialic acid oligosaccharides.

[0069] Based on a general inventive concept, Lh The preparation of Siase1 is consistent with the process of the method of the present invention described above. The specific steps of the preparation method can be referred to the above implementation scheme. Since the above use adopts some or all of the technical solutions of the above implementation scheme, it has at least all the beneficial effects brought about by the technical solutions of the above implementation scheme, which will not be repeated here.

[0070] The following describes preferred embodiments of the present invention, but the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0071] Example 1 Sialidase ( Lh Preparation of Siase1

[0072] Lactobacillus helsingborg Lactobacillus helsingborgensis The data is from the NCBI database, GenBank accession number: WP_109917843.1.

[0073] Sialidase ( Lh The gene sequence of Siase1 was constructed by Suzhou Hongxun Biotechnology Co., Ltd. using pET28a(+) and expressed using E. coli BL21(DE3).

[0074] Will contain genes LhRecombinant Escherichia coli BL21(DE3) of Siase1 was inoculated into LB medium and cultured at 37°C and 200 rpm until the OD 600 reached approximately 0.6-0.8. 0.1 mM IPTG was added to a final concentration, and the culture was induced at 20°C and 180 rpm for 24 h. The cells were collected by centrifugation, resuspended in 50 mM Tris-HCl (pH 7.4) buffer, and placed in an ice-water bath. The cells were sonicated for 10 min, centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The resulting pure enzyme solution was purified by nickel affinity chromatography.

[0075] The obtained Lh Siase1 was subjected to SDS-PAGE, and the results were as follows: Figure 1 As shown, Lh Siase1 has a molecular weight of approximately 39 kDa.

[0076] Example 2 Sialidase ( Lh Enzymatic property testing of Siase1

[0077] Enzyme activity assay: Using 1 mM 4MU-Neu5Ac as the substrate, 50 mM citrate-sodium citrate buffer (pH 5.5) and an appropriate amount of LhSiase1 were mixed on ice and reacted at 45 °C for 10 min. The reaction was then terminated by adding 1.9 mL of buffer containing 85 mmol / L glycine and 200 mmol / L sodium carbonate (pH 10.4). The fluorescence intensity of the released 4MU was measured at an excitation wavelength of 365 nm and an emission wavelength of 445 nm. A boiled sample was used as a control. The amount of enzyme required to release 1 μmol of 4MU per minute is defined as one enzyme activity unit (U).

[0078] Tested under different conditions Lh The enzyme activity of Siase1 is as follows:

[0079] Within a temperature range of 20 to 60°C, measurements were taken at 5°C intervals. Lh Siase1 enzyme activity, results as follows Figure 2 As shown, it indicates Lh The optimal reaction temperature for Siase1 is 45°C; further, the enzyme solution is incubated at 20–60°C for 30 min, cooled in an ice-water bath for 30 min, and then measured. Lh Siase1 enzyme activity, results as follows Figure 2 As shown, it can be seen Lh Siase1 remains stable below 40°C;

[0080] In different pH reaction systems, measurements were performed. Lh Siase1 enzyme activity, results as followsFigure 3 As shown, it indicates Lh The optimal pH for Siase1 is 5.0; the enzyme solution was incubated for 30 min in different pH reaction systems, then cooled in an ice-water bath for 30 min, and the results were measured. Lh Siase1 enzyme activity, results as follows Figure 3 As shown, it can be seen Lh Siase1 is stable in the pH range of 4.0-7.0;

[0081] Various metal ions and EDTA were added to a final concentration of 1 mM in different reaction systems (pH 3.0-6.0 citrate-sodium citrate buffer, pH 6.0-8.0 phosphate buffer, pH 7.0-9.0 Tris-HCl buffer, and pH 8.5-10.5 glycine-sodium hydroxide buffer, respectively). The results are as follows: Figure 4 As shown, it can be seen that metal ions and EDTA have an effect on... Lh Siase1 enzyme activity was not significantly affected.

[0082] Example 3 Preparation of a composition containing sialic acid oligosaccharides

[0083] Take 60 mg / mL lactose, 10 mg / mL bird's nest fragments (purchased from Taobao Mall, originating from Malaysia), and a final concentration of 0.4 U / mL. Lh Siase1 was reacted at 40℃ and pH 5.0 for 1 h. The reaction was terminated by boiling at 100℃ for 10 min, and the results were then analyzed by high performance liquid chromatography. The detection conditions were: Aminex HPX-87C column (4.6 x 250 mm), ultrapure water as the mobile phase, flow rate of 0.5 mL / min, temperature of 70℃, and sialic acid oligosaccharide standard as a control.

[0084] In this invention, bird's nest fragments are used as the substrate and lactose as the acceptor, through... Lh Following Siase1 catalysis, the reaction products, besides the remaining lactose acceptors, mainly consist of sialic acid oligosaccharides with concentrated molecular weights and single chromatographic peaks. Liquid chromatography results are as follows... Figure 5 As shown, the product spectrum is pure, indicating that the present invention has extremely high reaction specificity and effectively avoids the formation of complex by-products.

[0085] Furthermore, this invention greatly improves the bioavailability of sialic acid glycoproteins in bird's nest fragments, which have a large molecular weight (>100 kDa) and are difficult to absorb, by converting them into sialic acid oligosaccharides with a small molecular weight and high water solubility.

[0086] Example 4 Optimization of Reaction Conditions

[0087] Regarding the preparation method in Example 3, the amount of LhSiase1 enzyme, lactose concentration, concentration of bird's nest fragments, and reaction time were optimized as follows:

[0088] (1) Optimization of enzyme dosage: The final concentration of enzyme dosage should be selected to be between 0.1 U / mL and 1 U / mL.

[0089] (2) Optimization of lactose concentration: The final concentration of lactose was selected to be between 1w / v% and 10w / v%.

[0090] (3) Optimization of bird's nest fragment concentration: Select the final concentration of bird's nest fragments between 0.25w / v% and 2.5w / v%.

[0091] (4) Optimization of reaction time: The reaction time range is selected between 0 and 300 min.

[0092] The content of sialic acid oligosaccharides produced under different conditions was determined, and the results are as follows: Figure 6 As shown.

[0093] It can be seen that the optimal reaction conditions are: a final enzyme concentration of 0.4 U / mL; a final lactose concentration of 6 w / v; a final concentration of bird's nest fragments of 1.0 w / v; and a reaction time of 5 h.

[0094] Example 5 Different sialidases Lh Siase1 production capacity comparison

[0095] Choose from respectively Lactobacillus mulieris Sialidase LM Siase3 (WP_224783249.1), and derived from Kribbella sp. ALI-6-A sialidase Ks Siase2 (ONI68920.1), and the present invention Lh Siase1 was reacted under the same reaction conditions (60 mg / mL lactose, 10 mg / mL bird's nest fragments, 0.4 U / mL enzyme addition, 40℃, pH 5.0) for 2 hours. Previous tests under these conditions showed that... LM Siase3 and Ks The production of sialic acid oligosaccharides was carried out under the condition that Siase2 was also stable, and the results are shown in Table 1.

[0096] Table 1

[0097]

[0098] It can be seen that, Ks Siase2 can efficiently convert sialic acid glycoproteins into sialic acid monomers, but it lacks the ability to synthesize sialic acid oligosaccharides.LM Siase3 has transglycosylation activity in addition to hydrolyzing sialic acid monomers. However, under the same reaction conditions, Lh Siase1 catalyzes the synthesis of sialic acid oligosaccharides in Bird's Nest fragments at a content significantly higher than LM Siase3, indicating that the sialic acid oligosaccharide produced by the application has Lh Siase1 has incomparable advantages in terms of functional integrity and catalytic effect.

[0099] In addition, according to the liquid phase results, it can be found that, compared with the sialic acid oligosaccharide synthesized by LM Siase3, the product generated by the application is more pure, the main peak is single, the number of impurity peaks is less, and the by-products are less, which is conducive to its absorption in the human body. This indicates that Lh Siase1 is the best catalyst for realizing the high-value conversion of Bird's Nest fragments.

[0100] The above is only a preferred embodiment of the application, and is not intended to limit the protection scope of the application.

Claims

1. A method for preparing a composition containing sialic acid oligosaccharides, characterized in that, Includes the following steps: Grind the bird's nest into small pieces, then add lactose; join in Lh Siase1 was reacted, and after the reaction was completed, a composition containing sialic acid oligosaccharides was obtained. in, Lh The amino acid sequence of Siase1 is shown in SEQ ID NO.

1. Lh The final concentration of Siase1 is from 0.1 U / mL to 1 U / mL, the final concentration of lactose is from 1 w / v% to 10 w / v%, and the final concentration of bird's nest fragments is from 0.25 w / v% to 2.5 w / v.

2. The method according to claim 1, characterized in that, The Lh The final concentration of Siase1 was 0.3 U / mL to 0.5 U / mL.

3. The method according to claim 1, characterized in that, The final concentration of lactose is 4 w / v% to 7 w / v.

4. The method according to claim 1, characterized in that, The final concentration of the bird's nest fragments is 0.5w / v% to 1.5w / v%.

5. The method according to claim 1, characterized in that, The reaction temperature is between 35°C and 45°C, and the reaction time is between 0.5 h and 24 h.

6. The method according to claim 5, characterized in that, The reaction time is 1 to 5 hours.

7. The method according to claim 1, characterized in that, The bird's nest fragments are derived from the residue after bird's nest processing.

8. The amino acid sequence is as shown in SEQ ID NO.

1. Lh The use of Siase1 in the preparation of bird's nest powder preparation containing sialic acid oligosaccharides is characterized by, The bird's nest powder preparation is prepared by the following method. Grind the bird's nest into small pieces, then add lactose; add Lh Siase1 is reacted, and after the reaction is complete, the bird's nest powder preparation is obtained. in, Lh The final concentration of Siase1 was 0.1 U / mL to 1 U / mL.

9. The use according to claim 8, characterized in that, The final concentration of the lactose is from 1 w / v% to 10 w / v, and the final concentration of the bird's nest fragments is from 0.25 w / v% to 2.5 w / v.

10. The use according to claim 8, characterized in that, The reaction temperature is 35°C to 45°C, and the reaction time is 0.5h to 24h.

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