A method for preparing mogroside Ⅲ E by bioconversion of mogroside V

Abstract

The present application relates to the field of biological transformation of mogroside, and discloses a method for efficiently biotransforming mogroside V to prepare mogroside III E. A kind of plant endophytic fungus is used to biotransform mogroside V to obtain mogroside III E. The method is simple in operation, and the product has high conversion yield, so it can be used for large-scale preparation of mogroside III E. The method is simple, efficient, safe, green and low in cost, and provides a new method for industrial production, food production and new drug preparation.

Description

Technical Field

[0001] This invention relates to a method for preparing mogroside III E in the field of biotechnology. Background Technology

[0002] Monk fruit, native to China, is a plant with both edible and medicinal properties, widely used in traditional Chinese medicine and the food industry. Monk fruit saponins, due to their strong sweetness and lack of side effects, are often used as calorie-free natural sweeteners. Monk fruit saponin V is one of the main components of total monk fruit saponins, accounting for up to 52%. It is a complex tetracyclic triterpenoid saponin. In contrast, monk fruit saponin III E is a hydrolysis product of monk fruit saponin V. The difference in chemical structure results in monk fruit saponin III E exhibiting different characteristics in biological activity and pharmacological effects compared to monk fruit saponin V. Existing research has shown that monk fruit saponin III E has important pharmacological effects in anti-inflammation, alleviating gestational diabetes, acute lung injury, and pulmonary fibrosis, and shows great potential in the development of natural drugs. However, research on methods for converting monk fruit saponin V into monk fruit saponin III E is currently scarce, severely limiting the application and promotion of monk fruit saponin III E.

[0003] The structural differences among mogrosides mainly lie in the number and structure of sugar groups. Mogroside V contains multiple sugar groups, while mogroside III E is synthesized by hydrolysis, which removes the outer 1,6-glucose groups at positions C-3 and C-24. Although they share the same tetracyclic triterpenoid core structure, the difference in the number of sugar groups leads to significant differences in their physiological activities. Therefore, mogroside III E can be synthesized by hydrolyzing the sugar groups at positions C-3 and C-24 in the mogroside V molecule. Figure 1 ).

[0004] Previous studies have used chemical acid hydrolysis to prepare mogrosides with fewer sugar moss numbers from mogroside V. However, chemical acid hydrolysis requires stringent reaction conditions, and the hydrolysis products are complex and difficult to control. This method often results in low conversion yields, and subsequent product separation and purification are challenging. With the promotion of the green and low-carbon economy concept, biotransformation, as an environmentally friendly and efficient technology, is receiving increasing attention. Plant endophytic fungi, due to their ability to secrete various bioactive compounds and enzymes, have become a research hotspot in recent years. Compared with traditional chemical hydrolysis methods, biotransformation catalyzed by plant endophytic fungi is simple to operate, has mild reaction conditions, high specificity, high catalytic efficiency, low cost, and is environmentally friendly. Using plant endophytic fungal fermentation technology for the biotransformation of mogrosides can avoid the byproduct problems of acid hydrolysis and achieve stable and efficient preparation of mogrosides with fewer sugar moss numbers. Currently, there are few reports on the preparation of mogroside III and E through microbial transformation technology.

[0005] Based on this, the present invention employs plant endophytic fungal fermentation technology to successfully convert mogroside V into mogroside III E, providing a novel, green, and sustainable biotransformation method. Summary of the Invention

[0006] This invention discloses a highly efficient method for the biotransformation of mogroside V to prepare mogroside III E. The method utilizes endophytic fungi to biotransform mogroside V, yielding mogroside III E. The fungal strain used in this invention exhibits strong adaptability, the method is simple to operate, and the conversion yield is high, enabling large-scale production of mogroside III E. This method is simple, efficient, safe, environmentally friendly, and low-cost, making it suitable for applications in various fields such as industrial production, food production, and new drug development.

[0007] The technical solution adopted in this invention is as follows.

[0008] Plant endophytic fungus Chaetomium globosum ( Chaetomium globosum DX-THS3, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC No: M 201605, and deposited in May 2015.

[0009] The plant endophytic fungus *Chaetoceros* ( Chaetomium globosum The application of DX-THS3 in the production of mogroside III E through bioconversion is carried out according to the following steps.

[0010] The culture medium was prepared as follows: the primary medium consisted of potato dextrose agar (PDA), comprising 200 g / L potato, 20 g / L glucose, and 20 g / L agar powder. The fermentation medium was a modified version of the PDA medium, comprising 200 g / L potato, 20 g / L glucose, and 5 g / L mogroside V.

[0011] Strain activation, picking Chaetomium globulus ( Chaetomium globosum DX-THS3 mycelia were inoculated into the primary culture medium and incubated upside down at 28 ℃ for 7 days. The strain was activated when the white, fluffy mycelia covered the entire culture medium.

[0012] Biotransformation was performed by inoculating activated mycelia onto a fermentation medium and carrying out aerobic fermentation in a shaker at 28 ℃ and 160 rpm for 7 days. Fermentation broth samples were collected every 24 hours during fermentation, centrifuged at 4 ℃ and 7000 rpm for 10 minutes, and the supernatant was collected.

[0013] For preliminary detection by thin-layer chromatography (TLC), a small amount of supernatant was sampled using a capillary tube and spotted onto a TLC plate. The developing solvent was dichloromethane:methanol = 5:3 (v / v). After chromatography, a 10% ethanol sulfate solution was used as the colorimetric reagent, and the plate was baked at high temperature for color development.

[0014] Ultra-high performance liquid chromatography (UPLC) was used for accurate detection of substances produced by biotransformation. An Agilent 1290 column (USA) was employed, packed with octadecylsilane-bonded silica gel (Agilent, ZORBAX Eclipse Plus C18 1.8-Micron, USA). A small amount of the supernatant from the biotransformation was extracted with an equal volume of n-butanol, separating the upper n-butanol phase. The obtained n-butanol phase was placed in a rotary evaporator and evaporated to a powder. The powder was dissolved in 1.5 mL of liquid-grade methanol and filtered through a 0.25 μm organic filter before being packaged into a vial for UPLC detection. The mobile phase consisted of water (78%) as mobile phase A and acetonitrile (22%) as mobile phase B, at a flow rate of 0.45 mL / min, a column temperature of 40 ℃, and a detection wavelength of 203 nm, with isocratic elution for 15 min.

[0015] The product was purified by rotary evaporation to dry it into powder, yielding a dried powder containing mogroside III and E. Further purification through impurity removal steps yielded the final mogroside III and E product.

[0016] In the product purification process described above, removing impurities from the dried powder includes the following steps: dissolving the dried powder in solution A to obtain a liquid mixture containing mogroside III and E; adding solution B to the liquid mixture for extraction and separation; collecting the upper layer solution containing mogroside III and E; repeating the extraction and separation step three times; and drying the upper layer liquid using a rotary evaporator to obtain mogroside III and E.

[0017] Solution A and solution B are composed of ultrapure water and n-butanol, and the volume ratio of solution A (lower layer solution) to solution B is 1:1.

[0018] The application of plant endophytic fungi in the preparation of mogrosides IV A, IV E, and II is also within the scope of protection of this invention, such as the biotransformation of mogroside V by plant endophytic fungi to prepare mogrosides IV A and / or IV E and / or mogroside II, or one or more of them.

[0019] This invention proposes for the first time a method for the specific transformation of mogroside V using plant endophytic fungi to prepare mogroside III E. Given the significant therapeutic effects of mogroside III E in anti-inflammatory and diabetes-alleviating aspects, this method has the potential for widespread application in food additives, pharmaceutical preparation, and other fields. Furthermore, compared with traditional acid hydrolysis methods, this method offers advantages such as simplicity, efficiency, safety, and environmental friendliness, demonstrating significant potential for industrial application. Attached Figure Description

[0020] Figure 1 The reaction formula for the biotransformation of mogroside V into mogroside III E is given.

[0021] Figure 2 It is a plant endophytic fungus, *Chaetoceros*. Chaetomium globosum Colony morphology of DX-THS3.

[0022] Figure 3 This is a thin-layer chromatography image of mogroside III E obtained using the present invention.

[0023] Figure 4 The image shows the ultra-high performance liquid chromatogram of mogroside III E prepared using this invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Chaetomium coccidioides ( Chaetomium globosum DX-THS3 is a plant endophytic fungus that was isolated and screened from the stems of wild rice in Dongxiang by our laboratory and is now preserved at the China Collection Center for Classic Cultures of Wuhan University.

[0028] Example 1: Plant endophytic fungus Chaetomium globosum ( Chaetomium globosum Activation of DX-THS3.

[0029] *Chaetomium coccidioides* will be stored at -80℃. Chaetomium globosum DX-THS3 glycerol tube culture was inoculated onto primary culture medium and incubated upside down at 28 °C for 7 days.

[0030] The results are as follows Figure 2As shown, in the early stages of cultivation, the mycelium appears as white, fluffy hairs, growing loosely and radiating outwards. It is mostly aerial mycelium that adheres tightly to the surface of the culture medium. In the later stages of cultivation, the mycelium dries, turns grayish-white, and covers the entire surface of the culture medium.

[0031] Example 2: Utilizing the plant endophytic fungus *Chaetoceros globosum* ( Chaetomium globosum A method for preparing mogroside III E by biotransformation of DX-THS3.

[0032] The primary culture medium consisted of potato dextrose agar (PDA), comprising 200 g / L potato, 20 g / L glucose, and 20 g / L agar powder. The fermentation medium was a modified version of PDA medium, comprising 200 g / L potato, 20 g / L glucose, and 5 g / L mogroside V. Both medium components were dissolved in deionized water and sterilized at 121 °C for 20 minutes.

[0033] The activated Chaetomium globosum (from Example 1) Chaetomium globosum DX-THS3 was inoculated into a fermentation medium containing mogroside V and subjected to aerobic fermentation in a shaker at 28 ℃ and 160 rpm for 7 days. Samples were taken every 24 hours during the fermentation period. The supernatant was collected after centrifugation at 4 ℃ and 7000 rpm for 10 minutes.

[0034] Example 3: Detection method for mogroside III E prepared by biological methods.

[0035] Thin-layer chromatography and ultra-high performance liquid chromatography were used to detect biotransformation products, respectively.

[0036] (1) Thin-layer chromatography (TLC) detection of mogroside V and mogroside III E: A small amount of supernatant and standard were applied to a TLC plate (Qingdao Ocean Chemical Plant) using a capillary tube. After the TLC plate was air-dried, it was placed in a developing tank for chromatography. The developing solvent was dichloromethane:methanol = 5:3 (v / v). After chromatography, the TLC plate was removed and the solvent was evaporated. A 10% ethanol sulfate solution was used as the colorimetric reagent, which was sprayed evenly over the entire TLC plate. The plate was then baked at 105 °C for about 5 minutes for color development. The results are as follows: Figure 3 As shown, in a fermentation medium containing mogroside V, the plant endophytic fungus *Chaetoceros globosum* (…) Chaetomium globosum DX-THS3 can be hydrolyzed to produce mogroside III E.

[0037] (2) Detection of mogroside V and mogroside III E by ultra-high performance liquid chromatography (UHPLC): UHPLC: Agilent 1290; Column: Agilent ZORBAX Eclipse Plus C18 (2.1×50 mm, 1.8 μL); Loading volume: 2 μL; Mobile phase: A: water; B: acetonitrile; Program: 0-15 min, 22% B, 78% A; Column temperature: 40 ℃; Detector: UV detector, 203 nm; Flow rate: 0.45 mL / min. The standards were mogroside V and mogroside III E (Chengdu Manster).

[0038] The results are as follows Figure 4 As shown, after 7 days of biotransformation in a fermentation medium containing mogroside V, the plant endophytic fungus *Chaetomium globosum* (…) was successfully transformed. Chaetomium globosum DX-THS3 can be hydrolyzed to produce mogroside III E.

[0039] Example 4: Isolation and purification method for bio-preparation of mogroside III E.

[0040] Using the plant endophytic fungus *Chaetomium globosum* ( Chaetomium globosum The experiment to prepare mogroside III E by biotransformation of DX-THS3 was repeated three times, and the specific steps of each experiment are as follows.

[0041] The supernatant from Example 2 was rotary evaporated to 20 mL, pre-frozen at -70 °C, and then freeze-dried to obtain a dried powder of the conversion product. The conversion product was detected by UPLC detection method according to step (2) of Example 3 of the present invention, using mogroside V and mogroside III E as standards. Qualitative analysis was performed based on the retention time of the standards, and quantitative analysis was performed using the standard curve method (external standard method).

[0042] The dried powder of the conversion product was dissolved in a liquid consisting of n-butanol and water at a volume ratio of 1:1 (v / v). The upper liquid n-butanol phase was extracted and separated, and the process was repeated three times. The n-butanol phase was rotary evaporated until no organic solvent flowed out, pre-frozen at -70°C, and then freeze-dried to obtain pure mogroside III E.

Claims

1. The method for preparing mogroside III E involves using mogroside V as a substrate and undergoing biotransformation with plant endophytic fungi to obtain reactants containing mogroside III E.

2. The method according to claim 1, the biotransformation application of plant endophytic fungi, characterized in that, The Chaetomium globulus ( Chaetomium globosum Application of the DX-THS3 biotransformation method for the preparation of mogroside III E from mogroside V.

3. The method according to claim 1 or 2, characterized in that: 1) Picking out Chaetomium globulus ( Chaetomium globosum DX-THS3 was inoculated into primary culture medium and incubated upside down at 28 °C for 7 days to activate the strain; 2) The activated mycelium was inoculated into the fermentation medium and aerobic fermentation was carried out in a shaker at 28 ℃ and 160 rpm for 7 days to obtain the biotransformation product, mogroside III E.

4. The method according to claim 2, characterized in that: Thin-layer chromatography was used to detect biotransformation products. The developing solvent was dichloromethane:methanol = 5:3 (v / v). After chromatography, 10% ethanol sulfate solution was used as the colorimetric reagent, and the product was baked at high temperature for color development. Ultra-high performance liquid chromatography was used to detect biotransformation products. Water was used as mobile phase A (78%), acetonitrile was used as mobile phase B (22%), the flow rate was 0.45 mL / min, the column temperature was 40 ℃, the detection wavelength was 203 nm, and isocratic elution was performed for 15 min.

5. The method according to claim 2, characterized in that: The separation and purification process includes the following steps: centrifuging the fermentation broth to allow mogroside III E to enter the supernatant, collecting the supernatant to obtain a supernatant containing mogroside III E, drying the supernatant to obtain a dried powder containing mogroside III E, removing impurities from the dried powder to obtain mogroside III E.

6. The method according to claim 2, characterized in that: Removing impurities from the dried powder includes the following steps: dissolving the dried powder in solution A to obtain a liquid mixture containing mogroside III and E; adding solution B to the liquid mixture for extraction and separation; collecting the upper layer solution containing mogroside III and E; repeating the extraction and separation step three times; and drying the upper layer liquid using a rotary evaporator to obtain mogroside III and E; wherein solution A and solution B are composed of ultrapure water and n-butanol, and the volume ratio of solution A to solution B is 1:1.

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