The invention relates to 2apos; method for analyzing carbohydrates in fucosyllactose product

Abstract

The invention relates to the technical field of fermentation engineering, in particular to a method for analyzing carbohydrates in a 2 '-fucosyllactose product. The carbohydrate analysis method comprises the following steps: aiming at a 2 '-fucosyllactose product, separating carbohydrates through ion exchange chromatography, and the conditions are as follows: a mobile phase A comprises 4-6mM NaOH and 1-3mM NaAC; the mobile phase B is prepared from 80 to 120 mM of NaOH and 80 to 120 mM of NaAC; the elution procedure is a specific gradient elution procedure. According to the carbohydrate analysis method of the 2 '-fucosyllactose product, provided by the invention, miscellaneous sugar with relatively high similarity with the 2'-fucosyllactose in the 2 '-fucosyllactose product can be effectively separated, so that the analysis efficiency and accuracy of carbohydrates in the 2'-fucosyllactose product can be effectively improved; the method has important significance in the related production field of 2 '-fucosyllactose.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, and in particular to a method for ' -Analysis method of sugar substances in fucosyllactose products. Background Art

[0002] Fucosyllactose is the most abundant in HMO (human milk oligosaccharides) and has high nutritional and medical value in inhibiting pathogen infection, regulating intestinal flora, and enhancing immunity. However, fucosyllactose produced by extraction or chemical synthesis cannot meet its needs. Currently, microbial fermentation is a commonly used biotechnology method in the existing technology and has been commercialized. It has the characteristics of environmental friendliness and high efficiency. ' -The fucosyllactose fermentation broth contains target oligosaccharides, biomass, microbial cells, proteins, and other carbohydrates except the target oligosaccharides, of which 2 ' -Fucosyllactulose is 2 ' -Fucosyllactose is a common isomerized sugar in fermentation broth, and its presence affects the final quality of the product.

[0003] In the prior art, about 2 ' -Amide bonded chromatographic columns are usually used to separate fucosyllactose. ' -The various sugars in the fucosyllactose fermentation broth system were analyzed by high performance liquid chromatography-differential detection (HPLC-RID) and quantified by external standard method. Alternatively, hydrophilic retention chromatography column was used for separation, HPLC-RID analysis, and area normalization method for quantification. However, no matter which method was used, it failed to separate it from other sugars in the system, which would directly affect 2 ' -Fucosyllactose overall content analysis, thereby misleading the purification process and having an adverse effect on product production. In addition, 2 ' -The fucosyllactose fermentation broth system also includes a variety of sugar substances such as lactose, fucose, fucosylgalactose, etc. These different sugar substances have certain similarities in chemical structure, and it is significantly difficult to separate all of these sugar substances. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a 2 ' -Analysis method of sugar substances in fucosyllactose products.

[0005] There are many existing technologies for 2 ' - The analytical methods for fucosyllactose, such as the draft for comments and GRAS of relevant regulations, are given in the relevant analytical methods, but these methods are not applicable to 2 '- Fucosyllactose products (containing 2 ' - Fucosyllactose products, or 2 ' -fucosyllactose as the main component of semi-finished products) ' - The separation effect of each component is poor during the detection of fucosyllactose, and it is difficult to separate them simultaneously2 ' -Fucosyllactose products contain all sugars, and the peak shape of the target is poor. The present invention has established a new 2 ' - Fucosyllactose analysis method, which can achieve one-time analysis of 2 ' - The various sugar components in the fucosyllactose product can be clearly displayed 2 ' -The content distribution of fucosyllactose and other miscellaneous sugars significantly improves the separation efficiency of different sugar substances and can separate 2 ' -Fucosyllactulose was completely separated from the system.

[0006] In a first aspect, the present invention provides a method for analyzing carbohydrate substances in a 2'-fucosyllactose product, comprising: For 2'-fucosyllactose products, the sugars were separated by ion exchange chromatography under the following conditions: Mobile phase A includes: 4~6mM NaOH and 1~3mM NaAC; mobile phase B includes: 80~120mM NaOH and 80~120mM NaAC; The gradient elution program includes: 0-(27.5~28.5) minutes, mobile phase A is maintained at 100%, and mobile phase B is maintained at 0; (27.5~28.5)-(29.5~30.5) minutes, mobile phase A is 70~80%, mobile phase B is (20~30)%; (29.5~30.5)-(44~46) minutes, mobile phase A is 5~15%, mobile phase B is 85~95%; At (44~46)-(55~65) minutes, mobile phase A was 100%.

[0007] Furthermore, the mobile phase A is 4.5-5.5 mM NaOH and 1.8-2.2 mM NaAC; the mobile phase B is 95-105 mM NaOH and 95-105 mM NaAC.

[0008] Further, the gradient elution procedure comprises: 0 min, mobile phase A was 100%, mobile phase B was 0; 28 min, mobile phase A 100%, mobile phase B 0; 28.1 min, mobile phase A 80%, mobile phase B 20%; 30 min, mobile phase A 75%, mobile phase B 25%; 30.1 min, mobile phase A 10%, mobile phase B 90%; 45 min, mobile phase A was 10%, mobile phase B was 90%; 45.1 min, mobile phase A 100%, mobile phase B 0; 60 min, mobile phase A was 100%, mobile phase B was 0.

[0009] Furthermore, the microorganism is one or more of Escherichia coli, lactic acid bacteria, yeast, Bacillus or Pseudomonas.

[0010] Furthermore, the 2'-fucosyllactose product includes: a product with 2'-fucosyllactose as a main component; or A semi-finished product or finished product with 2'-fucosyllactose as the main component obtained by processing the fermentation product obtained by microbial fermentation.

[0011] The method for analyzing carbohydrate substances in 2'-fucosyllactose products of the present invention is widely applicable, that is, it can be used for analyzing semi-finished products (such as high-purity 2'-fucosyllactose semi-finished products obtained by purification of fermentation products obtained by microbial fermentation) or finished products in the production process of 2'-fucosyllactose products, and can also be used for analyzing existing products with 2'-fucosyllactose as the main component (such as food, milk powder or medicine, etc.) after pre-treatment.

[0012] Furthermore, the chromatographic column used in the ion exchange chromatography is a strong anion exchange chromatographic column, the column temperature is 30-32° C., and the flow rate is 1-1.5 mL / min.

[0013] Furthermore, after the sugar substances are separated by the ion exchange chromatography, they are analyzed by using a differential detector, an evaporative light scattering detector, an electrochemical detector or a mass spectrometer detector.

[0014] In a second aspect, the present invention provides a method for analyzing carbohydrate substances in an improved 2'-fucosyllactose product. ' -Application of fucosyllactose in separation efficiency.

[0015] The present invention has the following beneficial effects: The present invention provides a method for analyzing carbohydrate substances in 2'-fucosyllactose products, wherein 2'-fucosyllactose in the 2'-fucosyllactose products are analyzed by ion exchange chromatography with a specific mobile phase and a gradient elution program. '- Fucosyllactose and other similar sugars such as lactose, fucose, fucosyllgalactose (especially 2 ' -fucosyllactulose) can be distinguished, which can clearly show the content distribution of different sugar substances, which can significantly improve 2 ' - Fucosyllactose detection efficiency can be used for component identification and process optimization, improving 2 ' -Fucosyllactose purity provides guidance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is a chromatogram of the 2'-fucosyllactose product provided in Example 1 of the present invention.

[0018] Figure 2 This is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 1 of the present invention.

[0019] Figure 3 This is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 2 of the present invention.

[0020] Figure 4 This is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 3 of the present invention.

[0021] Figure 5 It is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 4 of the present invention.

[0022] Figure 6 This is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 5 of the present invention.

[0023] Figure 7 This is a chromatogram of the 2'-fucosyllactose preparation provided in Comparative Example 6 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. 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.

[0025] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0026] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources, for example: Example 1 This embodiment provides a method for analyzing carbohydrate substances in a 2'-fucosyllactose product, comprising the following process: 1. Raw materials The object of analysis of the present invention is 2 ' - Fucosyllactose products (mainly composed of 2 ' -fucosyllactose, other major impurities include fucose, lactose, 2 ' -fucosyllactulose, fucosyllgalactose, 3-FL, difucosyllactose (D-FL), etc., of which 2 ' -Fucosyllactulose and 2 ' -fucosyllactose has similar properties to 2 ' The preparation of 2'-fucosyllactose products can refer to patent CN202211385633.X. It is only an example. In fact, the method provided by the present invention is applicable to various products with 2'-fucosyllactose as the main component.

[0027] 2. Analysis methods The present invention adopts liquid chromatography to carry out 2 ' -Analysis of sugar substances in fucosyllactose products. Specifically, the sample was dissolved in water and analyzed using a strong anion exchange chromatography column (250 mm×4 mm) and a high performance anion exchange chromatography (HPAEC-PAD) equipped with a pulse current detector. The column temperature was 30°C and the flow rate was 1 mL / min. The mobile phase A used in the chromatography was 5 mM NaOH+2 mM NaAC; the mobile phase B was 100 mM NaOH +100 m MnaAC; the gradient elution process is shown in the following table: Table 1 Gradient elution process

[0028] The results are as follows Figure 1 As shown: Through the method provided in this embodiment, for 2 ' - The sugars in the fucosyllactose product can be separated to the best degree, on the one hand, 2 ' -Fucosyllactose production and purification process provides guidance, on the other hand, can also be used for containing 2 ' -Related testing of fucosyllactose products to evaluate whether the products are qualified.

[0029] Comparative Example 1 This comparative example provides a high performance liquid chromatography based on differential detection (HPLC-RID) for 2 ' -fucosyllactose products were analyzed, and the analysis object was the same as in Example 1. This method is a conventional detection method in the prior art. ' -2 in fucosyllactose products ' - The method for fucosyllactose (draft for comments) specifically uses an amide bonded chromatographic column, high performance liquid chromatography-differential detection analysis, and quantitative analysis using the area normalization method. The liquid chromatography conditions are as follows: Preparation method of mobile phase: Accurately weigh 582.8 g of acetonitrile, add appropriate amount of water to obtain 857.2 g of solution, and then add 10 mL of triethylamine.

[0030] The isocratic elution method was adopted, with a flow rate of 1 mL / min, a column temperature of 25°C, and an injection solution concentration of approximately 5 g / L.

[0031] The results are as follows Figure 2 As shown: It can be seen from the figure that this method cannot separate lactose and 2'-fucosyllactulose in the system.

[0032] Comparative Example 2 This comparative example provides a method for analyzing 2'-fucosyllactose products based on high-performance anion exchange chromatography (HPAEC-PAD) equipped with a pulse current detector. The analysis object is the same as that in Example 1. The method is a method for detecting 2'-fucosyllactose provided by GRAS, specifically using a strong anion exchange chromatography column for separation, using the HPAEC-PAD method for detection, and using the area normalization method for quantification. The conditions of the ion exchange chromatography are as follows: Column temperature: 35°C, flow rate: 1 mL / min; injection solution concentration is about 5 g / L, mobile phase A is: 100 mM NaOH, mobile phase B is 100 mM NaOH + 300 mM NaOAc, gradient elution, elution program is: 0-20 min, 99.7% mobile phase A, 0.3% mobile phase B; 20.1-30 min, 75% mobile phase A, 25% mobile phase B; 30.1~45min, 99.7% mobile phase A, 0.3% mobile phase B.

[0033] The results are as follows Figure 3 As shown in the figure, it can be seen that this method cannot separate 2'-fucosyllactose and 2'-fucosyllactulose in the system.

[0034] Comparative Example 3 The method used in this comparative example is the same as that in Example 1, except that the mobile phase and the gradient elution procedure are different. Specifically, the mobile phase A is 5 mM NaOH, the mobile phase B is 100 mM NaOH + 100 mM NaAC, and the gradient elution process is shown in the following table: Table 2 Gradient elution process

[0035] The results are as follows Figure 4 As shown in the figure, it can be seen that although 2'-fucosyllactose and other miscellaneous sugars in the system can be separated, the peak broadening phenomenon of 2'-fucosyllactose is too obvious and the peak shape is poor.

[0036] Comparative Example 4 The method used in this comparative example is the same as that in Example 1, except that the mobile phase and the gradient elution procedure are different. Specifically, the mobile phase A is 5 mM NaOH, the mobile phase B is 100 mM NaOH + 100 mM NaAC, and the gradient elution process is shown in the following table: Table 3 Gradient elution process

[0037] The results are as follows Figure 5 As shown, it can be seen from the figure that although the peak shape of 2'-fucosyllactose is improved compared with Comparative Example 3, 3'-fucosyllactose and fucosyl-D-galactose cannot be separated.

[0038] Comparative Example 5 The method used in this comparative example is the same as that in Example 1, except that the mobile phase and the gradient elution procedure are different. Specifically, the mobile phase A is 5 mM NaOH, the mobile phase B is 100 mM NaOH + 100 mM NaAC, and the gradient elution process is shown in the following table: Table 4 Eluent gradient elution table (Comparative Example 5)

[0039] The results are as follows Figure 6 As shown in the figure, it can be seen that this method has problems such as very low lactose response, poor symmetry of 2'-fucosyllactose peak shape, and inability to separate 3'-fucosyllactose and fucosyl-D-galactose.

[0040] Comparative Example 6 The method used in this comparative example is the same as that in Example 1, except that the mobile phase and the gradient elution procedure are different. Specifically, the mobile phase A is 5 mM NaOH + 2 mM NaAC, the mobile phase B is 100 mM NaOH + 100 mM NaAC, and the gradient elution process is shown in the following table: Table 4 Eluent gradient elution table (Comparative Example 5)

[0041] The results are as follows Figure 7 As shown, the resolution of 2'-fucosyllactose and lactose decreased significantly, and the peak shape of 2'-fucosyllactulose became worse.

[0042] In summary, even a small adjustment will significantly affect the separation effect of various sugar substances in 2'-fucosyllactose products. Compared with comparative examples 1-6, the detection method provided in the above Example 1 can significantly better separate various sugar substances in 2'-fucosyllactose, and the peak shape is better.

[0043] 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 analyzing carbohydrate substances in 2'-fucosyllactose products, characterized in that: include: For 2'-fucosyllactose products, saccharide substances are separated by ion exchange chromatography, wherein: Mobile phase A includes: 4~6mM NaOH and 1~3mM NaAC; mobile phase B includes: 80~120mM NaOH and 80~120mMNaAC; The gradient elution program includes: 0-(27.5~28.5) minutes, mobile phase A is maintained at 100%, and mobile phase B is maintained at 0; (27.5~28.5)-(29.5~30.5) minutes, mobile phase A is 70~80%, mobile phase B is (20~30)%; (29.5~30.5)-(44~46) minutes, mobile phase A is 5~15%, mobile phase B is 85~95%; At (44~46)-(55~65) minutes, mobile phase A was 100%.

2. The method for analyzing carbohydrate substances according to claim 1, characterized in that: The mobile phase A is 4.5-5.5 mM NaOH and 1.8-2.2 mM NaAC; the mobile phase B is 95-105 mM NaOH and 95-105 mM NaAC.

3. The method for analyzing carbohydrate substances according to claim 1 or 2, characterized in that: The gradient elution procedure includes: 0 min, mobile phase A was 100%, mobile phase B was 0; 28 min, mobile phase A 100%, mobile phase B 0; 28.1 min, mobile phase A 80%, mobile phase B 20%; 30 min, mobile phase A 75%, mobile phase B 25%; 30.1 min, mobile phase A 10%, mobile phase B 90%; 45 min, mobile phase A was 10%, mobile phase B was 90%; 45.1 min, mobile phase A 100%, mobile phase B 0; 60 min, mobile phase A was 100%, mobile phase B was 0.

4. The method for analyzing carbohydrate substances according to any one of claims 1 to 3, characterized in that: The microorganism is one or more of Escherichia coli, lactic acid bacteria, yeast, Bacillus or Pseudomonas.

5. The method for analyzing carbohydrate substances according to any one of claims 1 to 3, characterized in that: The 2'-fucosyllactose product includes: a product with 2'-fucosyllactose as the main component; or A semi-finished product or finished product with 2'-fucosyllactose as the main component obtained by processing the fermentation product obtained by microbial fermentation.

6. The method for analyzing carbohydrate substances according to any one of claims 1 to 5, characterized in that: The chromatographic column used in the ion exchange chromatography is a strong anion exchange chromatographic column, the column temperature is 30-32° C., and the flow rate is 1-1.5 mL / min.

7. The method for analyzing carbohydrate substances according to any one of claims 1 to 6, characterized in that: After the sugar substances are separated by the ion exchange chromatography, they are analyzed by using a differential detector, an evaporative light scattering detector, an electrochemical detector or a mass spectrometer detector.

8. The method for analyzing carbohydrate substances according to any one of claims 1 to 7 is used to improve the content of 2'-fucosyllactose in 2'-fucosyllactose products. ' -Application of fucosyllactose in separation efficiency.

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