Method for improving stability of N-acetylneuraminic acid aqueous solution

Abstract

The invention provides a method for improving the stability of an N-acetylneuraminic acid aqueous solution. The method comprises the step of adding nicotinamide into the N-acetylneuraminic acid aqueous solution. The nicotinamide is added into the N-acetylneuraminic acid aqueous solution, so that the stability of the N-acetylneuraminic acid aqueous solution can be improved, the solution is not prone to discoloration, and the attenuation of the content of N-acetylneuraminic acid is reduced. Compared with the existing method, the method disclosed by the invention can be simultaneously applied to the fields of food and cosmetics, and has a wider application range.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for improving the stability of an N-acetylneuraminic acid aqueous solution. Background Art

[0002] Sialic acid (SA) is a series of nine-carbon monosaccharide derivatives containing amino groups. It is the general name for all neuraminic acid and its derivatives. However, there is only one in the human body, namely N-acetylneuraminic acid (N-acetylneuraminicacid, Neu5Ac), which is usually directly called sialic acid, SA. SA has been proven to be involved in a variety of physiological processes in the body, such as cell recognition and signal transduction. It has physiological functions such as regulating the half-life of blood proteins, resisting various toxins, cell adhesion, glycoprotein cleavage protection, antioxidant properties, promoting brain development and improving memory. In recent years, it has been used in the fields of food, cosmetics, and medicine.

[0003] When SA is used in food, cosmetics, etc., SA must be dissolved in water in a certain proportion during the pretreatment process, and then subjected to high-temperature sterilization treatment. After that, the SA content in the aqueous solution must be kept stable during the shelf life. Usually, after high-temperature sterilization treatment, the SA content will decrease to a certain extent, and after a period of shelf storage, the SA content will decrease again. In severe cases, SA loses more than 50% during the entire process of the process and shelf life, and the color of the aqueous solution will become darker. Since the reduction in SA content during pretreatment and storage will affect the performance of its physiological effects, and the darkening of the color will cause sensory defects in the applied product, the prior art requires a method that can maintain good stability of SA during high-temperature pretreatment and shelf life.

[0004] CN108659095A proposes a method for stabilizing sialic acid content, which can stabilize the sialic acid content in the cell culture clarification harvest liquid and avoid the decline of sialic acid during the multiple cycle time of the next capture step operation. The method adopted is to use an alkaline reagent to adjust the pH value of the cell culture clarification harvest liquid ≥ 8.0, and the alkaline reagent is preferably Tris-HCl buffer. CN 109776626A and CN109771316A disclose a method for improving the stability of sialic acid aqueous solution and its application, wherein the method is to use a strong base and / or hydrochloric acid to adjust the pH of the aqueous solution containing sialic acid to 5-12. Although the method can effectively improve the stability of the sialic acid aqueous solution and is simple to operate, it is necessary to maintain the acidic environment in some actual application fields, which limits the scope of application of the method. CN 111067102A adds potassium sorbate to the SA aqueous solution to maintain the stability of the SA aqueous solution. Although this method is simple and feasible, it brings in non-natural ingredients after all, and is limited in application in some foods claimed to be zero preservatives and pure natural. CN 111035581A mixes chitosan with sialic acid solution to enhance the stability of sialic acid. After high-temperature sterilization, the content of sialic acid decreases slightly. The method is simple to operate and easy to apply in industrialization. However, the viscosity of the entire solution increases significantly after adding chitosan, which also limits the application to a certain extent. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for improving the stability of an aqueous solution of N-acetylneuraminic acid, which is a new strategy for maintaining good stability of N-acetylneuraminic acid during processing such as high-temperature pretreatment and shelf life.

[0006] In a first aspect, the present invention provides a method for improving the stability of an aqueous solution of N-acetylneuraminic acid, comprising the step of adding nicotinamide to the aqueous solution of N-acetylneuraminic acid.

[0007] Nicotinamide, also known as vitamin B3 or vitamin PP, belongs to the vitamin B family and is one of the 13 essential vitamins for the human body. Nicotinamide is a component of coenzyme I and coenzyme II. It plays a role in hydrogen transfer in human biological metabolism, promotes tissue respiration, biological oxidation process and metabolism, and is of great significance in maintaining the integrity of body tissues such as skin, digestive tract, heart system and nervous system. In medicine, it can also be used for skin diseases such as pellagra, stomatitis, liver disease and solar dermatitis. Nicotinamide is widely used in skin care products. It can reduce pigmentation by inhibiting the transport of melanosomes, and has the effects of repairing skin barrier, brightening skin tone and anti-oxidation.

[0008] Nicotinamide itself has general stability and is affected by factors such as pH value, temperature and light. However, the present invention unexpectedly discovered that adding nicotinamide to an aqueous solution of N-acetylneuraminic acid can improve the stability of the aqueous solution of N-acetylneuraminic acid, and the stability includes that the solution is not easy to change color and the N-acetylneuraminic acid content decays less.

[0009] In the method of the present invention, the pH does not have to be neutral to alkaline, and good stability can be achieved under relatively acidic conditions. Moreover, nicotinamide is a functional raw material, which is different from ferrous pyrosulfate auxiliary materials and potassium sorbate non-natural food additives. Nicotinamide is beneficial and harmless, and is easily accepted by consumers. Moreover, the addition of nicotinamide does not change the viscosity of the stock solution, avoiding the thickening of the system caused by the addition of chitosan, so that the application will not be limited by the viscosity. The method of the present invention is simple to operate and easy to apply in industrialization.

[0010] According to the method for improving the stability of an aqueous solution of N-acetylneuraminic acid provided by the present invention, in the aqueous solution after addition, the mass concentration of nicotinamide is not lower than the mass concentration of N-acetylneuraminic acid.

[0011] In the technical solution of the present invention, in order to obtain better stability of the N-acetylneuraminic acid aqueous solution, after addition, the mass concentration of nicotinamide is not lower than the mass concentration of N-acetylneuraminic acid, that is, in the aqueous solution after addition, the mass concentration of nicotinamide is more than 1 times the mass concentration of N-acetylneuraminic acid.

[0012] Taking into account the stability improvement effect and cost, in the aqueous solution after addition, the mass concentration of nicotinamide is 1-5 times the mass concentration of N-acetylneuraminic acid.

[0013] According to the method for improving the stability of an N-acetylneuraminic acid aqueous solution provided by the present invention, the mass concentration of N-acetylneuraminic acid in the N-acetylneuraminic acid aqueous solution is 0.01-5%.

[0014] When the content of N-acetylneuraminic acid is 0.01-5%, it can fully adapt to the practical application of N-acetylneuraminic acid in the fields of food, cosmetics, etc.

[0015] Preferably, and more commonly in practical applications, the mass concentration of N-acetylneuraminic acid in the N-acetylneuraminic acid aqueous solution is 0.01-0.5%.

[0016] Furthermore, when the mass concentration of N-acetylneuraminic acid in the N-acetylneuraminic acid aqueous solution is less than 0.2%, the mass concentration of nicotinamide in the aqueous solution after addition is 1-5 times the mass concentration of N-acetylneuraminic acid.

[0017] When the mass concentration of N-acetylneuraminic acid in the N-acetylneuraminic acid aqueous solution is 0.2% or more, the mass concentration of nicotinamide in the aqueous solution after addition is 2.5-5 times the mass concentration of N-acetylneuraminic acid.

[0018] That is to say, for different concentrations of N-acetylneuraminic acid aqueous solutions, in order to achieve a better stability effect, the dosage range of niacinamide to be added is different.

[0019] At the same time, the present invention also found that although nicotinamide itself is easily hydrolyzed into nicotinic acid under relatively acidic conditions, in the presence of N-acetylneuraminic acid, in some practical application examples obtained by the method of the present invention, the aqueous solution of N-acetylneuraminic acid and nicotinamide can inhibit the decomposition of nicotinamide into nicotinic acid under the influence of the secondary amine group of N-acetylneuraminic acid.

[0020] In the present invention, after adopting the method, the N-acetylneuraminic acid aqueous solution does not change color during processing and storage, and the loss rate of N-acetylneuraminic acid is small. Under preferred conditions, the loss rate of N-acetylneuraminic acid is less than 3%.

[0021] Specifically, during the processing, the product may be subjected to UHT treatment at 120-130° C. for 5-30 s, or subjected to high temperature sterilization at about 121° C. The storage condition is usually room temperature and protected from light.

[0022] In a second aspect, the present invention provides application of the above method for improving the stability of an aqueous solution of N-acetylneuraminic acid in the preparation of food, health products or cosmetics containing N-acetylneuraminic acid.

[0023] Furthermore, the food, health product or cosmetic is a liquid product, and its pH value may range from 3 to 8.

[0024] For example, the above method is used to prepare beauty products containing N-acetylneuraminic acid and nicotinamide (such as oral liquid and other internal products, or external products such as creams), which have good market prospects. Nicotinamide itself has a beauty effect, and N-acetylneuraminic acid also has a good performance in the beauty field. The two have different mechanisms of action. The combination of nicotinamide and N-acetylneuraminic acid has a better beauty effect, and the product will not change color during the shelf life to cause sensory defects, nor will it affect the efficacy due to the attenuation of active ingredients.

[0025] The present invention provides a method for improving the stability of an N-acetylneuraminic acid aqueous solution. By adding nicotinamide to the N-acetylneuraminic acid aqueous solution, the stability of the N-acetylneuraminic acid aqueous solution can be improved, including the solution being less likely to change color and reducing the attenuation of the N-acetylneuraminic acid content. Compared with the existing addition of non-natural food additives such as potassium sorbate, or ferrous pyrosulfate auxiliary materials, or chitosan components that change the consistency of the system, the method of the present invention is more natural and healthy, and has a wider range of applications. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0028] In the following examples, SA is the abbreviation of N-acetylneuraminic acid. The concentrations involved in the following examples are all mass percentage concentrations, w / w%, unless otherwise specified.

[0029] Experimental example

[0030] A certain amount of N-acetylneuraminic acid was weighed and added into ultrapure water to prepare N-acetylneuraminic acid aqueous solutions of different concentrations.

[0031] The above N-acetylneuraminic acid aqueous solution was taken, and different masses of nicotinamide and different comparative substances were added, as shown in Table 1, and UHT high temperature sterilization was adopted, that is, heating at 125°C for 30s, and then an accelerated stability test was carried out, and the condition was placed at 37°C for 1 month. The color change of each group and the attenuation of N-acetylneuraminic acid are shown in Table 1.

[0032] Whether the solution color becomes darker is observed with the naked eye, and a freshly prepared sample that has not been treated with high temperature or accelerated is used as a control. "-" represents that the color does not darken, and "+" represents that the color darkens. A colorimeter is used for analysis.

[0033] The SA content was determined by high performance liquid chromatography (HPLC): the instrument was Shimadzu Lc-15c; the detection column was Bio-Rad AMINEX HPX 87H Organic Analysis Column (300×7.8mm); the column temperature was 60°C; the mobile phase was 6mmol sulfuric acid; the flow rate was 0.6mL / min; and the detection wavelength was 210nm (all experimental data were repeated 3 times, and the results were the average of 3 times).

[0034] The calculation formula of SA attenuation is: attenuation = (accelerated sample content - initial content) × 100% / initial content.

[0035] The nicotinamide content was determined by high performance liquid chromatography (HPLC) in accordance with GB / T5009.197-2003.

[0036] The calculation formula of the attenuation amount of niacinamide is: attenuation amount = (accelerated sample content - initial content) × 100% / initial content.

[0037] Table 1

[0038]

[0039] From the above results, it can be seen that adding nicotinamide to the aqueous solution of N-acetylneuraminic acid can improve the problems of N-acetylneuraminic acid loss and solution discoloration. When the concentration of nicotinamide is 1-5 times that of N-acetylneuraminic acid, the improvement effect is better.

[0040] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0041] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the stability of an aqueous solution of N-acetylneuraminic acid, characterized in that: The method comprises the step of adding nicotinamide to the N-acetylneuraminic acid aqueous solution.

2. The method for improving the stability of N-acetylneuraminic acid aqueous solution according to claim 1, characterized in that: In the aqueous solution after addition, the mass concentration of nicotinamide is not lower than the mass concentration of N-acetylneuraminic acid.

3. The method for improving the stability of N-acetylneuraminic acid aqueous solution according to claim 2, characterized in that, In the aqueous solution after addition, the mass concentration of nicotinamide is 1-5 times the mass concentration of N-acetylneuraminic acid.

4. The method for improving the stability of an aqueous solution of N-acetylneuraminic acid according to any one of claims 1 to 3, characterized in that: In the N-acetylneuraminic acid aqueous solution, the mass concentration of N-acetylneuraminic acid is 0.01-5%.

5. The method for improving the stability of N-acetylneuraminic acid aqueous solution according to claim 4, characterized in that: In the N-acetylneuraminic acid aqueous solution, the mass concentration of N-acetylneuraminic acid is 0.01-0.5%.

6. The method for improving the stability of N-acetylneuraminic acid aqueous solution according to claim 5, characterized in that: In the N-acetylneuraminic acid aqueous solution, the mass concentration of N-acetylneuraminic acid is less than 0.2%. In the aqueous solution after addition, the mass concentration of nicotinamide is 1-5 times the mass concentration of N-acetylneuraminic acid.

7. The method for improving the stability of N-acetylneuraminic acid aqueous solution according to claim 5, characterized in that: In the N-acetylneuraminic acid aqueous solution, the mass concentration of N-acetylneuraminic acid is 0.2% or more, and in the aqueous solution after addition, the mass concentration of nicotinamide is 2.5-5 times the mass concentration of N-acetylneuraminic acid.

8. Use of the method for improving the stability of an aqueous solution of N-acetylneuraminic acid according to any one of claims 1 to 7 in the preparation of food, health products or cosmetics containing N-acetylneuraminic acid.

9. The use according to claim 8, characterized in that: The food, health product or cosmetic is a liquid product, and the pH value may range from 3 to 8.

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