A method for detecting related substances of ergothioneine produced by fermentation

Abstract

The present application belongs to the field of medicine and food raw material analysis, and particularly relates to a detection method of related substances of fermentation ergothioneine, which is detected by high performance liquid chromatography, with three-bond amido bonded silica gel as the filler, and with a phosphate solution and acetonitrile in a volume ratio of 20-30:80-70 as the mobile phase. The present application can simultaneously and accurately detect the related substances of methionine, sodium thiosulfate, histidine and histidine betaine in the fermentation ergothioneine by the specific mobile phase and the specific chromatographic column, and can also quantitatively detect the related substances, thereby solving the quality control problem of the ergothioneine fermentation liquor and the pure ergothioneine after separation and purification, and being beneficial to the quality control of the ergothioneine fermentation liquor or the fermentation ergothioneine product, and ensuring the product quality of the ergothioneine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and food raw material analysis, specifically relating to a method for detecting related substances in fermented ergothioneine production. Background Technology

[0002] Ergothioneine (2-mercapto-L-histidine trimethylammonium sulfate, EGT) is a compound discovered in 1909, initially found in the fungus *Claviceps purpurea*. The pure form is a white crystalline solid with good water solubility, with an upper limit of solubility of 0.9 mol / L at room temperature. It is quite stable at physiological pH and in strongly alkaline solutions. Ergothioneine serves as an important energy source and structural component. Furthermore, it acts as a regulator or effector, playing a crucial role in metabolic homeostasis, such as antioxidation, anti-inflammation, and cell protection. Due to its significant and unique biological functions, researchers have long studied its applications. Currently, it is mainly used as an antioxidant and skin protectant, and is also used in the treatment of cataracts and organ transplantation.

[0003] With the widespread use of ergothioneine in the food, pharmaceutical, and cosmetic industries, expanding its production is necessary. Currently, ergothioneine synthesis mainly involves chemical synthesis, extraction from fungal mycelia, and microbial fermentation. Chemical synthesis suffers from drawbacks such as long pathways, low conversion rates, and significant pollution. Natural extraction methods are limited by the low ergothioneine content in mycelia, hindering extraction efficiency. Conventional microbial fermentation is constrained by the host cell's regulatory system, limiting yields to gram-level processes, making large-scale production difficult. Utilizing genetically engineered bacteria for large-scale fermentation to produce active enzymes, which are secreted outside the bacteria and then catalyze the synthesis of ergothioneine using substrates, results in high substrate concentration, high ergothioneine yield, and relatively low cost, and is now widely used in ergothioneine production. However, in addition to ergothioneine, the in vitro enzyme catalysis process also generates structurally similar impurities. If these impurities are not removed during extraction and purification, they will affect the purity of the ergothioneine. To ensure the quality of ergothioneine, it is necessary to control the fermentation process and the relevant substances in the extracted and purified ergothioneine to ensure that the purity of the fermented ergothioneine meets the requirements and that the quality is guaranteed. Summary of the Invention

[0004] To effectively control the quality of ergothioneine produced during fermentation, this invention provides a method for detecting related substances in ergothioneine production. This method employs high-performance liquid chromatography (HPLC), and the specific steps are as follows:

[0005] The test sample solution and the reference solution were injected into the chromatograph separately. The chromatographic conditions were as follows: Column: triple bonded amide-bonded silica gel; Mobile phase: phosphate solution and acetonitrile in a volume ratio of 20-30:80-70.

[0006] The reference solution is an aqueous solution of ergothioneine reference standard and related substance reference standard.

[0007] Furthermore, the sample solution to be tested is an aqueous solution containing ergothioneine; the aqueous solution containing ergothioneine includes ergothioneine fermentation broth;

[0008] The ergothioneine fermentation broth is produced by inoculating recombinant ergothioneine-producing Escherichia coli into a fermentation medium and fermenting it.

[0009] Furthermore, the fermentation medium is formulated as follows: yeast extract 5-15 g / L, glycerol 10-60 g / L, phosphate 3-10 g / L, ammonium sulfate 1.0-5.0 g / L, anhydrous magnesium sulfate 0.1-3 g / L, calcium sulfate dihydrate 10-20 mg / L, manganese sulfate monohydrate 10-20 mg / L, calcium carbonate 10-30 g / L, histidine 5-25 g / L, methionine 30-50 g / L, sodium thiosulfate 5-40 g / L, polyether defoamer 0.1-1.2 g / L, with the remainder being water.

[0010] Furthermore, each 1 ml of the reference solution contains 0.1–0.5 mg of ergothioneine and related substances.

[0011] Furthermore, the chromatographic column is a Waters BEHAmide, with dimensions of 4.6*250mm and 5µm; the volume ratio of the phosphate solution to acetonitrile is 20-25:80-75, preferably 22:78.

[0012] Furthermore, the phosphate solution is an aqueous solution of dipotassium hydrogen phosphate with a concentration of 5–20 mmol / L and a pH value of 5.0–8.0, preferably with a concentration of 5 mmol / L and a pH value of 6.9.

[0013] Furthermore, the chromatographic conditions include a detection wavelength of 200–220 nm, an injection volume of 5–15 μL, a flow rate of 0.8–1.2 mL / min, and a column temperature of 30–40 °C. Preferably, the detection wavelength is 215 nm, the injection volume is 10 μL, the flow rate is 0.8–1.2 mL / min, and the column temperature is 30 °C.

[0014] Furthermore, the chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of the related substance chromatographic peak in the chromatogram of the reference solution, indicating that the ergothioneine to be tested contains the corresponding related substance.

[0015] Furthermore, the content of the relevant substances is calculated by peak area using the external standard method.

[0016] Furthermore, the relevant substances are methionine, sodium thiosulfate, histidine, and / or histidine betaine.

[0017] This invention, through preliminary experiments, discovered that ergothioneine produced by fermentation generates unique process impurities during the fermentation process. These impurities all exhibit ultraviolet absorption. Therefore, a high-performance liquid chromatography (HPLC) method was developed for the analysis of related substances. The following is information on the relevant impurities:

[0018]

[0019] This invention provides a method for detecting ergothioneine-related substances using high-performance liquid chromatography (HPLC). Through a specific mobile phase and with the aid of a specific chromatographic column, it can simultaneously and accurately detect methionine, sodium thiosulfate, histidine, and histidine betaine in ergothioneine. It can also quantitatively detect these related substances, thereby solving the quality control problem of ergothioneine fermentation broth and its purified product. This method is beneficial for quality control of ergothioneine fermentation broth or ergothioneine products, ensuring the quality of ergothioneine products.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 Chromatograms of typical standard solutions at a flow rate of 0.8 ml / min during method validation;

[0023] Figure 2 Chromatogram of fermentation broth at a flow rate of 0.8 ml / min during method validation;

[0024] Figure 3 Chromatogram of a typical standard solution at a flow rate of 1.0 ml / min during method validation;

[0025] Figure 4 Chromatogram of fermentation broth at a flow rate of 1.0 ml / min during method validation;

[0026] Figure 5 Chromatogram of a typical standard solution at a flow rate of 1.2 ml / min during method validation;

[0027] Figure 6 Chromatogram of fermentation broth at a flow rate of 1.2 ml / min during method validation. Detailed Implementation

[0028] The reagents and equipment used in the specific embodiments of this invention were all purchased commercially. The ergothioneine fermentation broth used as the test sample was prepared by inoculating recombinant ergothioneine-producing Escherichia coli into a fermentation medium and fermenting it.

[0029] The recombinant Escherichia coli that produces ergothionein is E. coli W3110, which expresses N-methyltransferase Egt1, cysteine ​​desulfurase Egt2, cystathionine γ-lyase Cys3 and cystathionine β-synthetase Cys4, and was specifically provided by Shanghai Ruikang Biotechnology R&D Co., Ltd.

[0030] The fermentation medium is formulated as follows: yeast extract 5-15 g / L, glycerol 10-60 g / L, phosphate 3-10 g / L, ammonium sulfate 1.0-5.0 g / L, anhydrous magnesium sulfate 0.1-3 g / L, calcium sulfate dihydrate 10-20 mg / L, manganese sulfate monohydrate 10-20 mg / L, calcium carbonate 10-30 g / L, histidine 5-25 g / L, methionine 30-50 g / L, sodium thiosulfate 5-40 g / L, polyether defoamer 0.1-1.2 g / L, with the remainder being water. During fermentation, 30% sodium hydroxide is used as a pH adjuster to maintain the pH value at 7.0-7.2.

[0031] Example 1: HPLC Detection of Ergothionein Produced by Fermentation in this Invention

[0032] 1) Preparation of reference solution

[0033] Accurately weigh 15 mg each of ergothioneine, methionine, sodium thiosulfate, histidine, and histidine betaine reference standards (accurate to 0.1 mg), place them in a 100 mL volumetric flask, add ultrapure water to dilute to the mark, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the final product.

[0034] 2) Inject ergothioneine fermentation broth and reference solution separately into the high-performance liquid chromatograph; chromatographic conditions are as follows:

[0035] Chromatographic column: Waters BEHAmide (4.6*250mm, 5um)

[0036] Mobile phase: 5 mmol / dipoxat hydrogen phosphate (pH = 6.90): acetonitrile = 22:78

[0037] Detection wavelength: 215nm

[0038] Flow rate: 0.8 ml / min

[0039] Column temperature: 30℃

[0040] Injection volume: 10 μL

[0041] 4) The chromatogram of the test solution shows a chromatographic peak with the retention time of the related substance chromatographic peak in the chromatogram of the reference standard, that is, ergothioneine contains the corresponding related substance; the content of the related substance is calculated by the external standard method based on the peak area.

[0042] The following experimental examples further illustrate the beneficial effects of the present invention:

[0043] Experimental Example 1: Methodological Validation of the Invention

[0044] 1. Methodological Validation ①

[0045] Instrument: Agilent 1260 high performance liquid chromatograph

[0046] Chromatographic column: Waters BEHAmide (4.6*250mm, 5um)

[0047] Mobile phase: 5 mmol / dipoxat hydrogen phosphate (pH = 6.90): acetonitrile = 22:78

[0048] Detection wavelength: 215nm

[0049] Flow rate: 0.8 ml / min

[0050] Column temperature: 30℃

[0051] Injection volume: 10 μL

[0052] Solution preparation: Accurately weigh 15 mg each of ergothioneine, methionine, sodium thiosulfate, histidine, and histidine betaine reference standards (accurate to 0.1 mg), place them in a 100 mL volumetric flask, dilute to the mark with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate in a sample vial for analysis by high performance liquid chromatography (HPLC). Parallel measurements should be performed simultaneously, and the relative deviation (RD) of the response values ​​(peak area / concentration) of parallel measurements should be ≤1.0%.

[0053] Chromatograms of typical standard solutions of each impurity and main component, determined under the above chromatographic conditions, are shown below. Figure 1 The chromatogram of the fermentation broth under this condition is shown in [reference needed]. Figure 2 The separation and recovery rates of each impurity peak met the requirements. See Table 1 below.

[0054] Table 1. Methodological Validation Results

[0055]

[0056]

[0057] Note: The recovery rate in the validation project was tested on the fermentation broth, while the other indicators were tested on the reference solution. Validation should be performed in accordance with the requirements of the "Guidelines for Analytical Method Validation" in the Chinese Pharmacopoeia. The same applies below.

[0058] 2. Methodological Validation ②

[0059] Instrument: Agilent 1260 high performance liquid chromatograph

[0060] Chromatographic column: Waters BEH Amide (4.6*250mm, 5µm)

[0061] Mobile phase: 5 mmol / dipoxat hydrogen phosphate (pH = 6.90): acetonitrile = 22:78

[0062] Detection wavelength: 215nm

[0063] Flow rate: 1.0 ml / min

[0064] Column temperature: 30℃

[0065] Injection volume: 10 μL

[0066] Solution preparation: Accurately weigh 15 mg each of ergothioneine, methionine, sodium thiosulfate, histidine, and histidine betaine reference standards (accurate to 0.1 mg), place them in a 100 mL volumetric flask, dilute to the mark with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate in a sample vial for analysis by high performance liquid chromatography (HPLC). Parallel measurements should be performed simultaneously, and the relative deviation (RD) of the response values ​​(peak area / concentration) of parallel measurements should be ≤1.0%.

[0067] Chromatograms of typical standard solutions of each impurity and main component, determined under the above chromatographic conditions, are shown below. Figure 3 The chromatogram of the fermentation broth under this condition is shown in [reference needed]. Figure 4 The resolution and recovery rate of each impurity peak met the requirements. The detection limit concentration of each impurity and the main component was less than 0.02% of the sample concentration, and the quantitation limit concentration was less than 0.05% of the sample concentration. See Table 2 below.

[0068] Table 2 Methodological Validation Results

[0069]

[0070]

[0071] 3. Methodological Validation ③

[0072] Instrument: Agilent 1260 high performance liquid chromatograph

[0073] Chromatographic column: Waters BEHAmide (4.6*250mm, 5um)

[0074] Mobile phase: 5 mmol / dipoxat hydrogen phosphate (pH = 6.90): acetonitrile = 22:78

[0075] Detection wavelength: 215nm

[0076] Flow rate: 1.2 ml / min

[0077] Column temperature: 30℃

[0078] Injection volume: 10 μL

[0079] Solution preparation: Accurately weigh 15 mg each of ergothioneine, methionine, sodium thiosulfate, histidine, and histidine betaine reference standards (accurate to 0.1 mg), place them in a 100 mL volumetric flask, dilute to the mark with ultrapure water, shake well, filter through a 0.22 μm microporous membrane, discard the initial filtrate, and collect the subsequent filtrate in a sample vial for analysis by high performance liquid chromatography (HPLC). Parallel measurements should be performed simultaneously, and the relative deviation (RD) of the response values ​​(peak area / concentration) of parallel measurements should be ≤1.0%.

[0080] Chromatograms of typical standard solutions of each impurity and main component, determined under the above chromatographic conditions, are shown below. Figure 5 The chromatogram of the fermentation broth under this condition is shown in [reference needed]. Figure 6 The separation and recovery rates of each impurity peak met the requirements. The detection limit concentrations of each impurity and the main component were less than 0.02% of the sample concentration, and the quantitation limit concentrations were less than 0.05% of the sample concentration. See Table 3 below.

[0081] Table 3. Methodological Validation Results

[0082]

[0083]

[0084] The above validation results show that, under the chromatographic parameters (Waters BEH Amide column, 4.6*250mm, 5µm, mobile phase 22:78 of 5mmol / dipoxetine hydrogen phosphate and acetonitrile, detection wavelength 215nm, flow rate 0.8–1.2ml / min, column temperature 30℃), the method for determining ergothionein-related substances produced by fermentation is highly specific, sensitive, and accurate.

[0085] In summary, this invention, through a specific mobile phase and a specific chromatographic column, can simultaneously and accurately detect impurities such as methionine, sodium thiosulfate, histidine, and histidine betaine in ergothioneine, and can also quantitatively detect impurities in ergothioneine, thereby ensuring the quality of fermented ergothioneine products and possessing practical application value.

Claims

1. A method for detecting ergothioneine-related substances, characterized in that: It is detected using high-performance liquid chromatography (HPLC), and the specific steps are as follows: Inject the test sample solution and the reference solution into the chromatograph separately, and apply the following chromatographic conditions: Column: packed with triple-bonded amide-bonded silica gel; mobile phase: phosphate solution and acetonitrile in a volume ratio of 20-30:80-70. The reference solution is an aqueous solution of ergothioneine reference standard and related substance reference standard.

2. The detection method as described in claim 1, characterized in that: The sample solution to be tested is an aqueous solution containing ergothioneine; the aqueous solution containing ergothioneine includes ergothioneine fermentation broth; The ergothioneine fermentation broth is produced by inoculating recombinant ergothioneine-producing Escherichia coli into a fermentation medium and fermenting it.

3. The detection method as described in claim 1 or 2, characterized in that: The fermentation medium was formulated as follows: yeast extract 5–15 g / L, glycerol 10–60 g / L, phosphate 3–10 g / L, ammonium sulfate 1.0–5.0 g / L, anhydrous magnesium sulfate 0.1–3 g / L, calcium sulfate dihydrate 10–20 mg / L, manganese sulfate monohydrate 10–20 mg / L, calcium carbonate 10–30 g / L, histidine 5–25 g / L, methionine 30–50 g / L, sodium thiosulfate 5–40 g / L, polyether defoamer 0.1–1.2 g / L, with the remainder being water.

4. The detection method as described in claim 1, characterized in that: The reference solution contains 0.1–0.5 mg of ergothioneine and related substances per 1 ml.

5. The detection method as described in claim 1, characterized in that: The chromatographic column is a Waters BEH Amide, with dimensions of 4.6*250mm and a thickness of 5µm; the volume ratio of the phosphate solution to acetonitrile is 20-25:80-75, preferably 22:

78.

6. The detection method as described in claim 5, characterized in that: The phosphate solution is a dipotassium hydrogen phosphate aqueous solution with a concentration of 5–20 mmol / L and a pH value of 5.0–8.0, preferably with a concentration of 5 mmol / L and a pH value of 6.

9.

7. The detection method as described in claim 1, characterized in that: The chromatographic conditions are as follows: detection wavelength is 200–220 nm, injection volume is 5–15 μL, flow rate is 0.8–1.2 mL / min, column temperature is 30–40 °C, preferably detection wavelength is 215 nm, injection volume is 10 μL, flow rate is 0.8–1.2 mL / min, and column temperature is 30 °C.

8. The detection method as described in claim 1, characterized in that: The chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of the related substance chromatographic peak in the chromatogram of the reference solution, indicating that the ergothioneine to be tested contains the corresponding related substance.

9. The detection method as described in claim 1, characterized in that: The content of the relevant substances is calculated by peak area using the external standard method.

10. The detection method as described in claim 1, 4, or 9, characterized in that: The relevant substances are methionine, sodium thiosulfate, histidine, and / or histidine betaine.

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