A method for extracting sialic acid based on subcritical water extraction

Abstract

The application discloses a sialic acid extraction method based on subcritical water extraction and belongs to the technical field of sialic acid extraction. The extraction method comprises the following steps: S1, preparing a yolk solution; S2, subcritical water reaction extraction of a sialic acid solution; S3, vacuum concentration; S4, resin adsorption purification; and S5, concentration and drying. The sialic acid product obtained by the sialic acid extraction method based on subcritical water extraction has an average purity of more than 98%; while the purity is maintained, the recovery rate is greatly improved and reaches more than 94%; the whole process can be completed in only 10-12 hours, the time is greatly shortened, the efficiency is improved, the labor cost is reduced, and the problem of a large amount of strong acid waste liquid is avoided.

Description

Technical Field

[0001] This invention relates to the field of sialic acid extraction technology, and in particular to a sialic acid extraction method based on subcritical water extraction. Background Technology

[0002] Sialic acid is an important component of gangliosides and polysialic acid chains that modify nerve cell adhesion molecules. It plays a vital role in promoting infant brain and nervous system development, enhancing infant cognitive abilities, fighting viruses, and promoting intestinal development. The main sources of sialic acid for the body are synthesis by the liver and exogenous food. However, because the liver's ability to synthesize sialic acid is limited in infants, and the rapid brain development of infants (especially premature infants) places extremely high demands on dietary nutrients, sufficient exogenous sialic acid intake must be ensured in the early stages of life to meet the body's developmental needs.

[0003] Breast milk (or the mother's own milk) is the primary source of nutrients for infants (or fetuses), and its abundant sialic acid is extremely important for promoting the growth and development of the fetus in late pregnancy or the infant after birth. Therefore, sialic acid products are an essential ingredient in infant formula. In addition, sialic acid also has anti-recognition effects, antiviral effects, inhibits leukocyte adhesion, and anti-inflammatory effects.

[0004] Given the continued attention given to the application of sialic acid in infant and maternal foods, developing efficient, safe, and economical industrial production methods for sialic acid is of significant economic and social importance. Currently, the main methods for preparing sialic acid include chemical synthesis, enzymatic synthesis, microbial fermentation, and extraction from natural raw materials.

[0005] Among them, enzymatic synthesis and chemical synthesis both require expensive substrates N-acetylglucosamine and pyruvate, resulting in high costs; chemical synthesis also requires a large amount of organic solvents and the synthesis conditions are relatively harsh, making it difficult to be widely used in industrial-scale production; microbial fermentation produces low product concentrations and is often accompanied by byproducts; whole-cell biotransformation uses living cells, which also presents problems of catabolism and secondary products during the transformation process.

[0006] Natural extraction methods, which separate and extract nutrients from food raw materials, offer the advantages of safety and reliability. Poultry egg yolks are widely available, inexpensive, and rich in functional components. In particular, the sialic acid in poultry eggs is Neu5Ac, which has the same structure as sialic acid in breast milk, making it an ideal raw material for preparing sialic acid suitable for infant formula.

[0007] However, due to the low content of sialic acid in egg yolks, improper separation and preparation methods result in significant losses and loss of activity. Therefore, obtaining high-quality sialic acid has become one of the research challenges. Existing egg yolk sialic acid extraction technologies generally involve steps such as defatting with organic reagents, phenol pretreatment or acidification, column chromatography decolorization or desalting, and ion exchange purification. These methods generally suffer from practical problems such as chemical reagent contamination, cumbersome procedures, high time, energy consumption, and cost, severe pollution, and unstable sialic acid product quality.

[0008] Furthermore, each step in the separation process inevitably leads to varying degrees of sialic acid loss; simultaneously, the numerous steps result in a very low product yield, only around 50-60%. Therefore, developing a new technology for large-scale production of sialic acid that meets the safety requirements of infant formula, is simple to operate, has a short purification cycle, and achieves high yield will not only enrich the variety of functional ingredients required for infant formula but also have significant application value in increasing the added value of poultry eggs and optimizing the structure of the poultry egg industry. Summary of the Invention

[0009] This invention provides a sialic acid extraction method based on subcritical water extraction, aiming to solve the problems of chemical reagent pollution, long production cycle, high energy consumption and cost, and serious pollution that are common in existing sialic acid preparation technologies.

[0010] To achieve the above objectives, the present invention provides a method for sialic acid extraction based on subcritical water extraction, wherein S1, an egg yolk solution is prepared;

[0011] S2. Sialic acid in the egg yolk solution obtained in step S1 is extracted by subcritical water reaction. After the reaction is completed, solid-liquid separation is performed and the supernatant is collected.

[0012] S3. Concentrate the supernatant obtained in step S2 under reduced pressure to 1 / 8 to 1 / 5 of its original volume.

[0013] S4. The concentrated solution obtained in step S3 is purified by resin adsorption, and the collected chromatographic peaks are desalted by nanofiltration to obtain sialic acid solution.

[0014] S5. The sialic acid solution obtained in step S4 is concentrated and dried to obtain pure sialic acid.

[0015] Preferably, the preparation of the egg yolk solution in step S1 includes: uniformly adding a certain volume of distilled water to the egg yolk raw material.

[0016] Preferably, the egg yolk raw material is fresh poultry egg yolk, and the amount of distilled water added is 0.6 to 3 times the volume of the fresh poultry egg yolk.

[0017] Preferably, the egg yolk raw material is egg yolk powder, and the amount of distilled water added is 8 to 20 times the volume of the egg yolk powder.

[0018] Preferably, the subcritical water reaction extraction in step S2 includes:

[0019] The egg yolk solution was placed in a high-pressure reactor, sealed, and filled with nitrogen. Subcritical water was prepared under conditions of 0–20 MPa and 100–170 °C. The subcritical water was then used for extraction for 5–40 min.

[0020] Preferably, the reduced pressure concentration in step S3 is as follows: the supernatant is placed in a rotary evaporator and concentrated at 0.09 MPa and 75°C.

[0021] Preferably, the resin adsorption purification in step S4 includes: using a gel-type macroporous adsorption resin as a chromatography packing material, injecting the supernatant obtained in step S3 into the resin, eluting with 0-5 mol / L sodium chloride at isocratic concentration, and collecting the chromatographic peaks.

[0022] Preferably, the macroporous adsorption resin is a gel-type basic anion exchange resin, the ionic strength of the eluent is 0.1-0.5 M, and the elution flow rate is 0-3 mL / min.

[0023] Preferably, the sialic acid extraction method based on subcritical water extraction according to claim 1 is characterized in that the nanofiltration conditions in step S4 are: using an NF90 membrane module, performing membrane filtration at 20-40°C and an operating pressure of 20-40 bar, and collecting the retentate.

[0024] Therefore, the present invention employs the above-mentioned subcritical water extraction-based sialic acid extraction method, which has the following technical advantages:

[0025] (1) Based on the composition characteristics of egg yolk, a sialic acid extraction scheme was designed: First, based on the differences in polarity of lipids, fat-soluble pigments and sialic acid molecules, they were separated by subcritical water method. Then, taking advantage of the huge difference in charge properties between sialic acid and other coexisting protein molecules, anion exchange resin adsorption method was used to purify them. The hydrolysis of sialic acid and decolorization of egg yolk were achieved simultaneously by subcritical water method. The whole process only requires one step, which greatly simplifies the operation steps and also reduces the cost of purchasing chromatography packing materials.

[0026] (2) The entire process of this invention can be completed in only 10 to 12 hours, which is significantly shorter than the 3 to 5 days cycle of the existing method, thus improving efficiency and reducing labor costs.

[0027] (3) This invention uses subcritical water method to prepare sialic acid from egg yolk. By controlling the temperature and time of subcritical water reaction, the dissolution rate of sialic acid is improved and the degradation of sialic acid is reduced. The purity of the obtained sialic acid sample reaches more than 98%. While maintaining high purity, the recovery rate is greatly improved, from about 60% of the existing method to more than 90%, making full use of poultry egg resources.

[0028] (4) The sialic acid preparation process of the present invention uses only water as the sole extractant and does not use any organic reagents or acid or alkali solutions, which effectively avoids the acid water discharge and environmental pollution problems caused by conventional technical methods, maximizes the preservation of the biological activity of sialic acid products, and the prepared sialic acid products are highly safe and suitable for addition to infant formula milk powder.

[0029] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0030] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0031] Figure 1 This is a flowchart illustrating the separation and preparation of sialic acid in an embodiment of the present invention;

[0032] Figure 2 The following are elution patterns of sialic acid on adsorption resin in the embodiments of the present invention, wherein (a) is the elution pattern of sialic acid on adsorption resin in Example 1, (b) is the elution pattern of sialic acid on adsorption resin in Example 2, (c) is the elution pattern of sialic acid on adsorption resin in Example 3, and (d) is the elution pattern of sialic acid on adsorption resin in Example 4.

[0033] Figure 3 The images show high-performance liquid chromatograms of sialic acid used in the experiments. Part (a) is the blank spectrum of the solvent peak, part (b) is the spectrum of the sialic acid standard, part (c) is the spectrum of the sialic acid sample prepared in Example 1, part (d) is the spectrum of the sialic acid sample prepared in Example 2, part (e) is the spectrum of the sialic acid sample prepared in Example 3, and part (f) is the spectrum of the sialic acid sample prepared in Example 4. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0036] Example 1

[0037] A method for sialic acid extraction based on subcritical water extraction includes the following steps:

[0038] S1. Preparation of egg yolk powder reducing solution

[0039] Take 20g of egg yolk powder, slowly add 200ml of distilled water, and stir thoroughly to make an egg yolk solution.

[0040] S2, subcritical water extraction and decolorization

[0041] The egg yolk solution was placed in a high-pressure reactor, sealed, and then purged with N2 for about 10 minutes to remove air from the reactor. The reactor was then reacted at 150°C for 20 minutes. After the reaction was completed, solid-liquid separation was performed, and the supernatant was collected.

[0042] S3, Reduced Pressure Concentration

[0043] The resulting supernatant was placed in a rotary evaporator and concentrated to 1 / 5 of its original volume at 0.09 MPa and 70 °C for further purification.

[0044] S4. Resin adsorption purification

[0045] Using 201×4 as the chromatography column packing material, the column was equilibrated with distilled water after packing. The supernatant obtained in step S3 was injected into the column, and isocratic elution was performed with 0.5M NaCl. The elution solution containing sialic acid was collected. The elution curve is shown below. Figure 2 As shown in Figure a.

[0046] S5. The sialic acid-containing elution solution obtained in step S4 is subjected to nanofiltration membrane removal of NaCl using an NF90 membrane module at 20°C and an operating pressure of 35 bar to obtain a concentrated solution containing sialic acid. After drying, pure sialic acid is obtained.

[0047] Example 2

[0048] A method for sialic acid extraction based on subcritical water extraction includes the following steps:

[0049] S1. Preparation of egg yolk liquid

[0050] Take a fresh egg, separate the egg white and yolk, collect 20mL of yolk, and slowly add 120mL of distilled water to the yolk while stirring. After adding the water, continue stirring for 2.5 hours to obtain a fresh egg yolk solution.

[0051] S2, Subcritical Water Extraction

[0052] The fresh egg yolk solution was placed in a high-pressure reactor, sealed, and then purged with N2 for about 10 minutes to remove air from the reactor. The reactor was then reacted at 130°C for 20 minutes. After the reaction was completed, solid-liquid separation was performed, and the supernatant was collected.

[0053] S3, Reduced Pressure Concentration

[0054] The supernatant was placed in a rotary evaporator and concentrated to 1 / 5 of its original volume at 0.09 MPa and 70°C for further purification.

[0055] S4, Resin Adsorption Purification

[0056] Using a 201×4 column as the chromatography packing material, the column was equilibrated with distilled water after packing. The concentrated supernatant obtained in step S3 was injected into the column, and isocratic elution was performed with 0.5M NaCl. The elution solution containing sialic acid was collected. The elution curve is shown below. Figure 2 As shown in b.

[0057] S5. The sialic acid-containing elution solution obtained in step S4 is subjected to nanofiltration membrane removal of NaCl using an NF90 membrane module at 20°C and an operating pressure of 35 bar to obtain a concentrated solution containing sialic acid. After drying, pure sialic acid is obtained.

[0058] Example 3

[0059] A method for sialic acid extraction based on subcritical water extraction includes the following steps:

[0060] S1, Duck egg yolk solution

[0061] Take a fresh duck egg, separate the egg white and yolk, collect 20mL of yolk, and slowly add 120mL of distilled water to the duck egg yolk while stirring. After adding the water, continue stirring for 2.5 hours to obtain a fresh egg yolk solution.

[0062] S2, Subcritical Water Extraction

[0063] The fresh egg yolk solution was placed in a high-pressure reactor, sealed, and then purged with N2 for about 10 minutes to remove air from the reactor. The reactor was then reacted at 130°C for 20 minutes. After the reaction was completed, solid-liquid separation was performed, and the supernatant was collected.

[0064] S3, Reduced Pressure Concentration

[0065] The supernatant obtained in step S2 was placed in a rotary evaporator and concentrated to 1 / 5 of its original volume at 0.09 MPa and 70°C for further purification.

[0066] S4, Resin Adsorption Purification

[0067] Type 335 resin was used as the chromatography packing material. After packing, the chromatography column was equilibrated with distilled water. The concentrated supernatant obtained in step S3 was injected into the chromatography column, and isocratic elution was performed with 0.5M NaCl. The eluent containing sialic acid was collected. The elution curve is shown below. Figure 2 As shown in c.

[0068] S5. The sialic acid-containing elution solution obtained in step S4 is subjected to nanofiltration membrane removal of NaCl using an NF90 membrane module at 20°C and an operating pressure of 35 bar to obtain a concentrated solution containing sialic acid. After drying, pure sialic acid is obtained.

[0069] Example 4

[0070] A method for sialic acid extraction based on subcritical water extraction includes the following steps:

[0071] S1, Quail egg yolk liquid

[0072] Take fresh quail eggs, separate the egg white and yolk, collect 10mL of yolk, and slowly add 50mL of distilled water to the yolk while stirring. After adding the water, continue stirring for 2.5 hours to obtain a fresh egg yolk solution.

[0073] S2, subcritical water extraction and decolorization

[0074] The fresh egg yolk solution obtained in step S1 was placed in a high-pressure reactor, sealed, and then purged with N2 for about 10 minutes to remove the air from the reactor. The reactor was then reacted at 130°C for 20 minutes. After the reaction was completed, solid-liquid separation was performed, and the supernatant was collected.

[0075] S3, Reduced Pressure Concentration

[0076] The supernatant obtained in step S2 was placed in a rotary evaporator and concentrated to 1 / 5 of its original volume at 0.09 MPa and 70°C for further purification.

[0077] S4, Resin Adsorption Purification

[0078] Using 335 as the chromatography packing material, the column was equilibrated with distilled water after packing. The supernatant obtained in step S3 was injected into the column, and isocratic elution was performed with 0.5M NaCl. The elution solution containing sialic acid was collected. The elution curve is shown below. Figure 2 As shown in d.

[0079] S5. The sialic acid-containing elution solution obtained in step S4 is subjected to nanofiltration membrane removal of NaCl using an NF90 membrane module at 20°C and an operating pressure of 35 bar to obtain a concentrated solution containing sialic acid. After drying, pure sialic acid is obtained.

[0080] Test

[0081] (1) Purity determination

[0082] The determination was performed by high performance liquid chromatography (HPLC). An EclipsePlus C18 column (4.6 × 250 mm) was used for analysis. The liquid chromatography system was Waters 1525, the detection wavelength was 230 nm, the mobile phase was acetonitrile:water (9:1), and the flow rate was 1 mL / min.

[0083] Using solvent peak signals ( Figure 3 After removing the background peaks from the solvent in section a), the retention time corresponding to the sialic acid standard is then used as a reference. Figure 3 b) The signal peak of sialic acid in the sample is determined, and the peak area is integrated by the system's built-in software to quantify the sample purity.

[0084] The high-performance liquid chromatograms of the sialic acid samples prepared in Examples 1 to 4 are shown below. Figure 3 As shown in Figures c to f, it is clear that the liquid phase elution spectra of the sialic acid samples obtained in Examples 1 to 4 of this invention are consistent with those of the standard.

[0085] The peak area was integrated using software, and the sample purity was quantified based on the peak area. The calculated purities for Examples 1 to 4 were 98.32%, 97.84%, 98.01%, and 98.20%, respectively, with an average of 98.09%.

[0086] (2) Recovery rate calculation

[0087] Based on the concentration, volume, and purity of sialic acid purified from egg yolk, the recovery rate of sialic acid in each example was calculated using the following formula:

[0088]

[0089] In Example 1, 20g of egg yolk powder was separated, purified, and dried to obtain sialic acid-containing powder. The obtained sample was reconstituted with deionized water to 200mL and then its content was determined. The sialic acid content of the sample was determined to be 90μg / mL using a colorimetric method. Based on this, the mass of pure sialic acid was calculated to be 18mg. The theoretical sialic acid content in egg yolk is 0.095%, and the theoretical mass of total sialic acid in 20g of egg yolk is 19mg. Therefore, the recovery rate of sialic acid in egg yolk in Example 1 can be calculated to be 94.73%.

[0090] The recovery rates of Examples 2 to 4 were calculated using the same method, and the results were 94.73%, 95.42%, 94.91%, and 94.01%, respectively. The average recovery rate of Examples 1 to 4 was 94.76%. The sialic acid recovery rate in the examples of this invention is much higher than the recovery rate of about 60% in the prior art.

[0091] Therefore, the present invention adopts the above-mentioned subcritical water extraction method for sialic acid extraction, and the average purity of the obtained sialic acid product reaches more than 98%; while maintaining high purity, the recovery rate is greatly improved, reaching more than 94%; the entire process of this method can be completed in only 10 to 12 hours, which greatly shortens the time, improves efficiency and reduces labor costs, and avoids the problem of generating a large amount of highly acidic waste liquid.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting sialic acid based on subcritical water extraction, characterized by, Includes the following steps: S1. Preparation of egg yolk solution: Add 0.6 to 3 times the volume of distilled water to fresh egg yolks and stir. S2. The egg yolk solution obtained in step S1 was extracted with subcritical water to obtain sialic acid. After the reaction was completed, solid-liquid separation was performed and the supernatant was collected. S3. Concentrate the supernatant obtained in step S2 under reduced pressure to 1 / 8 to 1 / 5 of its original volume; S4. The concentrated solution obtained in step S3 is purified by resin adsorption, and the collected chromatographic peaks are desalted by nanofiltration to obtain a sialic acid solution. S5. The sialic acid solution obtained in step S4 is concentrated and dried to obtain pure sialic acid. The subcritical water extraction method in step S2 is as follows: The egg yolk solution was placed in a high-pressure reactor, sealed, and filled with nitrogen. Subcritical water was prepared at 20 MPa and 100-170 °C. Subcritical water was then used for extraction for 5-40 min. The resin adsorption purification method in step S4 is as follows: using a gel-type macroporous adsorption resin as a chromatography packing material, the supernatant obtained in step S3 is injected into the resin, isocratic elution is performed with 0.5 mol / L sodium chloride, and the chromatography peaks are collected.

2. The method of claim 1, wherein the method is a subcritical water extraction-based extraction method of sialic acid. The fresh poultry egg yolks can be replaced with egg yolk powder, and the amount of distilled water added is 8 to 20 times the volume of the egg yolk powder.

3. The method of claim 1, wherein the subcritical water extraction is performed at a temperature of 150-350°C and a pressure of 4-25 MPa. The vacuum concentration method in step S3 is as follows: the supernatant is placed in a rotary evaporator and concentrated at 0.09 MPa and 75°C.

4. The method of claim 1, wherein the subcritical water extraction-based extraction of sialic acid is characterized by, The macroporous adsorption resin is a gel-type basic anion exchange resin.

5. The method of claim 1, wherein the subcritical water extraction-based extraction of sialic acid is characterized by, The nanofiltration conditions in step S4 are as follows: using an NF90 membrane module, performing membrane filtration at 20~40℃ and an operating pressure of 20~40 bar, and collecting the retentate.

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