A method for extracting mogroside V

Abstract

This invention discloses a method for extracting mogroside V, relating to the field of biotechnology. The method involves culturing, fermenting, centrifuging, and homogenizing *E. coli* containing glycosyltransferases UGT-MS1 and UGT-MS2 to obtain an enzyme supernatant. Then, mogroside IIE, uridine diphosphate glucose, the enzyme supernatant, and salt are added to a phosphate buffer solution for enzymatic catalysis to obtain a conversion solution. Finally, the solution is ultrafiltered, loaded onto a column, eluted, and then passed through an alkaline anion exchange resin. The effluent is concentrated and dried to obtain mogroside V. The product has a high yield and is environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for extracting mogroside V. Background Technology

[0002] Monk fruit (Siraiti agrosvenorii) is the dried, ripe fruit of the perennial herbaceous vine *Siraiti agrosvenorii*, belonging to the Cucurbitaceae family. Also known as *Lahan Guo*, *Shenxian Guo*, and *Guangguo Mubie*, it was among the first batch of medicinal and edible herbs in my country. my country has a long history of cultivating monk fruit, primarily in the shady areas of southern China, including Guangxi, Jiangxi, and Guizhou. As a traditional Chinese medicine, not only the fruit but also the roots, leaves, and flowers have medicinal value. In traditional Chinese medicine, its functions include clearing the lungs and throat, resolving phlegm and relieving cough, and moistening the intestines and promoting bowel movements. Due to its cooling and fluid-generating properties, it is very effective in treating bronchitis, sore throat, and phlegm. Modern pharmacological studies have shown that monk fruit also possesses anti-inflammatory, anti-tumor, and antioxidant effects. Although plant-derived natural products are widely recognized in today's market and have broad application prospects, research on the effective pharmacologically active substances in monk fruit is still in its initial stages. As a food, monk fruit is rich in vitamins, amino acids, and lipids, which can promote the development of the body. In addition, monk fruit is very sweet and low in calories, making it an excellent sugar substitute.

[0003] Mogrosides are the main terpenoid active substances in monk fruit, belonging to the cucurbitane-type tetracyclic triterpenoid saponins. They are also the main sweeteners in monk fruit, with a sweetness approximately 300 times that of sucrose. In mature fruit, mogroside V is the main sweetener, while a small amount of mogroside IV is also present. This sweet mogroside is widely used in the market and is called mogroside sweetener. In immature fruit, mogrosides II and III are the main components; at this stage, the fruit has no sweetness and may even have a strong bitter taste. In 2010, monk fruit extract received GRAS (Generally Recognized As Safe) certification from the Food and Drug Administration (FDA) and was approved as a dietary supplement and natural sweetener. Because mogrosides cannot be digested and absorbed by the human body, have a high sweetness, and are a non-sugar natural product, they can be added to cakes, bread, and other foods as a low-calorie, high-intensity sweetener to replace sucrose. They do not cause a spike in blood sugar after consumption, bringing benefits to obese and diabetic patients. With widespread social attention, many food and beverage companies have added mogrosides to their products in recent years, and the demand for mogrosides is expected to continue to grow. Furthermore, research on mogroside extracts in traditional Chinese medicine is becoming increasingly extensive. Due to the growing demand for healthy, low-sugar lifestyles, mogrosides are widely used in pharmaceutical throat lozenges and calorie-free health supplements.

[0004] Currently, the main method for obtaining mogrosides is plant extraction. However, this method is heavily influenced by the season and the quality of the mogroside fruit. Furthermore, the quality of the mogrosides varies, resulting in lower content and greater differences among the components. This leads to significant manpower and resources being required for separation and purification, and limitations imposed by different separation methods. Most importantly, the long ripening cycle of mogrosides means that the annual yield is far from meeting the ever-increasing demand. The other two methods for obtaining mogrosides are chemical synthesis and enzymatic hydrolysis. Chemical synthesis is extremely difficult, requiring multiple chemical protection and deprotection processes with very low catalytic efficiency and high energy consumption. The use of various chemical reagents during synthesis also causes severe environmental pollution. Enzymatic hydrolysis also faces significant challenges. Firstly, it suffers from poor stereoselectivity, making it difficult to identify specific sites for hydrolysis. Secondly, the degree of hydrolysis is difficult to control, making it impossible to obtain specific products with high sweetness and activity. Although enzymatic hydrolysis is fast and pollution-free, the yield of mogrosides remains very low. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for extracting mogroside V with high yield and environmental friendliness, which addresses the shortcomings of the existing technology.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0007] A method for extracting mogroside V includes the following steps:

[0008] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting culture broth was then fermented to obtain a fermentation broth. The nucleotide sequence of UGT-MS1 is shown in SEQ ID NO.1, and the nucleotide sequence of UGT-MS2 is shown in SEQ ID NO.2.

[0009] B: Centrifuge the fermentation broth to collect the bacterial cells, and then resuspend the bacterial cells in phosphate buffer solution to obtain a resuspended solution;

[0010] C: The obtained resuspension was homogenized and then centrifuged to obtain the enzyme clear solution;

[0011] D: Add mogroside IIE, uridine diphosphate glucose, enzyme solution, and salt to phosphate buffer solution, stir at 50-220 rpm to obtain a mixed solution, and carry out the enzyme catalytic reaction under stirring to obtain a conversion solution containing mogroside V.

[0012] E: Heat the conversion solution containing mogroside V to 80-90℃, keep it at this temperature for 20-40 minutes, then add calcium chloride and diatomaceous earth, and then filter to obtain the filtrate;

[0013] F: Pass the obtained filtrate through an ultrafiltration membrane and collect the supernatant from the ultrafiltration membrane;

[0014] G: Then adjust the pH of the obtained ultrafiltration membrane supernatant to 7.0-8.0, and then load it onto the column. Stop loading the column when the eluent has a sweet taste. After stopping the column loading, wash the column with deionized water until it is neutral, and then elute with 70% ethanol aqueous solution. Collect the eluent and then wash the column with water until there is no alcohol smell.

[0015] H: The obtained eluent is then fed into an alkaline anion exchange resin at a rate of 1-2 BV / h, and the effluent is collected;

[0016] I: The collected effluent is concentrated to a solid content of 30-50% w / w, and then spray-dried to obtain mogroside V.

[0017] Preferably, in step A, the culture medium is inoculated into the fermentation medium at an inoculum size of 5–10% v / v, the initial pH of the fermentation medium is 7.0, and the initial airflow rate is 0.5 m³ / h. 3 The fermentation process was carried out at a concentration of 0.05 MPa, a pressure of 35–38°C, and a rotation speed of 250–300 rpm. During fermentation, the stirring speed was automatically coupled to the dissolved oxygen level. The temperature was automatically controlled by the fermenter. The pH was maintained by adding 50% ammonia solution. The dissolved oxygen (DO) value was kept at 20–30% for the first 24 hours of fermentation. The culture was continued until the OD value of the bacterial culture reached a certain level. 600 The pH value was 30-60. IPTG was added to induce fermentation at a final concentration of 0.4% wt, and the mixture was fermented at a constant temperature of 30℃ for 24 hours to obtain the fermentation broth.

[0018] Preferably, in step B, the OD value of the resuspension is maintained at 140-160, the concentration of the phosphate buffer solution is 50-100 μM, and the amount of phosphate buffer solution added is 2-4 times the weight of the bacterial body.

[0019] Preferably, the homogenizer in step C operates under the following conditions: 40-100 MPa, temperature <37°C, and 2-4 treatments.

[0020] Preferably, in step D, the concentration of mogroside IIE is 100-200 μM; the concentration of uridine diphosphate glucose is 50-100 μM; the salt is one or more of calcium chloride and magnesium chloride, with a salt concentration of 50-100 μM; and the pH of the phosphate buffer solution is 6-8.

[0021] Preferably, the temperature of the enzyme-catalyzed reaction in step D is 20-45°C, the reaction time is 1-5 h, the amount of UGT-MS1 added is 5-10 U / ml, and the amount of UGT-MS2 added is 4-8 U / ml.

[0022] Preferably, in step E, the amount of calcium chloride added is 2-4% of the volume of the conversion liquid, and the amount of diatomaceous earth added is 3-5% of the volume of the conversion liquid.

[0023] Preferably, the pore size of the ultrafiltration membrane in step F is 5000-10000 Da.

[0024] Preferably, the infeed flow rate of the ultrafiltration membrane supernatant in step G is 0.5-1.0 BV / h;

[0025] The feed flow rate of deionized water is 0.5-1.5 BV / h;

[0026] Preferably, the column is washed with deionized water until neutral, and then subjected to gradient washing with 1.5-3 column volumes of 20-60% ethanol aqueous solution.

[0027] Preferably, the concentration conditions are a temperature of 60-80℃ and a vacuum degree of <-0.090Mpa;

[0028] The conditions for spray drying are an inlet air temperature ≤180℃ and an outlet air temperature of 80-100℃.

[0029] UGT-MS1 nucleotide sequence (SEQ ID NO.1)

[0030]

[0031] UGT-MS2 nucleotide sequence (SEQ ID NO.2)

[0032]

[0033] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0034] 1. Using calcium chloride as a flocculant is relatively inexpensive. It reacts with the phosphates present in the conversion liquid to form calcium phosphate precipitate, which can act as a filter aid during the filtration process, accelerate the filtration speed of the filtrate, and also achieve the effect of phosphorus removal, reducing the cost of subsequent wastewater treatment and being environmentally friendly.

[0035] 2. Passing the obtained filtrate through an ultrafiltration membrane can remove large protein molecules, thereby increasing the injection volume and separation efficiency of the resin in the later stages.

[0036] 3. Using an anion exchange resin suitable for mogroside V, the mogroside V product changes from its original pale yellow or brownish-yellow to white. Moreover, the eluent from the macroporous resin does not need to be concentrated and recovered with ethanol and can be directly loaded onto the column, shortening the production steps and production cycle. Attached Figure Description

[0037] Figure 1 This is the liquid chromatogram of mogroside V in Example 3. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to the embodiments.

[0039] Example 1

[0040] A method for extracting mogroside V includes the following steps:

[0041] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting bacterial cultures were inoculated into fermentation medium at an inoculum size of 5% v / v. The initial pH of the fermentation medium was 7.0, and the initial airflow rate was 0.5 m³ / h. 3 Fermentation was carried out at a pressure of 0.05 MPa, a fermentation temperature of 35°C, and a stirring speed of 250 rpm. During fermentation, the stirring speed was automatically coupled to the dissolved oxygen level. The temperature was automatically controlled by the fermenter. The pH was maintained by adding 50% ammonia solution. The dissolved oxygen (DO) value was kept at 20% for the first 24 hours of fermentation. The culture was continued until the OD value of the bacterial culture reached a certain level. 600 The value was 30, and IPTG was added at a final concentration of 0.4% wt to induce fermentation. The mixture was then fermented at a constant temperature of 30℃ for 24 hours to obtain the fermentation broth.

[0042] B: Centrifuge the fermentation broth to collect the cells, and then resuspend the cells in a 50 μM phosphate buffer solution to obtain a resuspended solution. The OD value of the resuspended solution is maintained at 140-160, and the phosphate buffer solution is twice the weight of the cells.

[0043] C: The obtained resuspension was homogenized and then centrifuged to obtain enzyme clear solution (enzyme activity of UGT-MS1 was 35 U / ml and enzyme activity of UGT-MS2 was 40 U / ml); the homogenization conditions were 40 MPa, temperature <37℃, and the treatment was repeated 4 times.

[0044] D: 2400g of mogroside IIE, 852g of uridine diphosphate glucose, 4.3L of UGT-MS1, 3.0L of UGT-MS2, and 166g of calcium chloride were added to 28.0L of phosphate buffer solution. The mixture was stirred at 50 rpm to obtain a homogeneous solution. An enzymatic reaction was then carried out under stirring to obtain 31.5L of conversion solution containing mogroside V. The concentrations of mogroside IIE, uridine diphosphate glucose, and salt in the mixture were 100 μm, 50 μm, and 50 μm, respectively. The pH of the phosphate buffer solution was 6. The enzyme catalytic reaction was carried out at 20°C for 5 hours. The dosage of UGT-MS1 was 5 U / ml of the mixture, and the dosage of UGT-MS2 was 4 U / ml.

[0045] E: Heat the conversion solution containing mogroside V to 80°C and keep it at this temperature for 20 minutes. Then add calcium chloride (2% of the volume of the conversion solution) and diatomaceous earth (3% of the volume of the conversion solution). Then filter to obtain 34.0 L of filtrate.

[0046] F: The obtained filtrate is passed through an ultrafiltration membrane, and 49.0 L of the ultrafiltration membrane supernatant is collected. The pore size of the ultrafiltration membrane is 5000 Da, and its purpose is to remove large protein molecules.

[0047] G: Then adjust the pH of the obtained ultrafiltration membrane supernatant to 7.0, and then load it onto a macroporous adsorption resin column, model LX-28. Stop loading the column when the effluent has a sweet taste. After stopping the loading, wash the column with deionized water at 0.5 BV / h until neutral. Then perform gradient washing with 1.5 column volumes of 20-60% ethanol aqueous solution. Then elute with 70% ethanol aqueous solution and collect 60.0 L of the eluent. Then wash with water until the column has no alcohol odor. The column feed flow rate is 0.5 BV / h throughout.

[0048] H: The obtained eluent was then fed into an alkaline anion exchange resin at a rate of 1 BV / h, and 75.0 L of effluent was collected.

[0049] I: The collected effluent was concentrated at a temperature of 60℃ and a vacuum degree of <-0.090Mpa until the solid content was 30%, and then spray-dried to obtain mogroside V, 3100g, with a purity of 98.8%. The spray-drying conditions were: inlet air temperature ≤180℃ and outlet air temperature 80-100℃.

[0050] Example 2

[0051] A method for extracting mogroside V includes the following steps:

[0052] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting bacterial cultures were inoculated into fermentation medium at an inoculum size of 6% v / v. The initial pH of the fermentation medium was 7.0, and the initial airflow rate was 0.5 m³ / h. 3 Fermentation was carried out at a concentration of / L, maintaining a pressure of 0.05 MPa, a fermentation temperature of 36℃, and a stirring speed of 270 rpm. During fermentation, the stirring speed was automatically coupled to the dissolved oxygen level. The temperature was automatically controlled by the fermenter. The pH was maintained by adding 50% ammonia water. The dissolved oxygen (DO) value was kept at 25% for the first 24 hours of fermentation. The culture was continued until the OD value of the bacterial culture reached a certain level. 600 The value was 40, and IPTG was added at a final concentration of 0.4% wt to induce fermentation. The mixture was then fermented at a constant temperature of 30℃ for 24 hours to obtain the fermentation broth.

[0053] B: Centrifuge the fermentation broth to collect the cells, and then resuspend the cells in a 60 μM phosphate buffer solution to obtain a resuspended solution. The OD value of the resuspended solution is maintained at 140-160, and the phosphate buffer solution is 3 times the weight of the cells.

[0054] C: The obtained resuspension was homogenized and then centrifuged to obtain enzyme clear solution ((the enzyme activity of UGT-MS1 was 36 U / ml and the enzyme activity of UGT-MS2 was 42 U / ml)); the homogenization conditions were 60 MPa, temperature < 37℃, and the treatment was repeated 3 times.

[0055] D: 3600g of mogroside IIE, 1192g of uridine diphosphate glucose, 7.0L of UGT-MS1, 4.0L of UGT-MS2, and 233g of calcium chloride were added to 16.0L of phosphate buffer solution. The mixture was stirred at 100rpm to obtain a homogeneous solution. An enzymatic reaction was then carried out under stirring to obtain 32.0L of conversion solution containing mogroside V. The concentrations of mogroside IIE, uridine diphosphate glucose, and salt in the mixture were 150 μm, 70 μm, and 70 μm. The pH of the phosphate buffer solution was 7. The enzyme catalytic reaction was carried out at 30℃ for 3 hours. The dosage of UGT-MS1 was 8 U / ml of the mixture, and the dosage of UGT-MS2 was 6 U / ml.

[0056] E: Heat the conversion solution containing mogroside V to 85°C and keep it at this temperature for 30 minutes. Then add calcium chloride (3% of the volume of the conversion solution) and diatomaceous earth (4% of the volume of the conversion solution), and then filter to obtain 34.5 L of filtrate.

[0057] F: The obtained filtrate is passed through an ultrafiltration membrane, and 50.5 L of the ultrafiltration membrane supernatant is collected. The pore size of the ultrafiltration membrane is 8000 Da, and its purpose is to remove large protein molecules.

[0058] G: Then, adjust the pH of the obtained ultrafiltration membrane supernatant to 8.0, and then load it onto a macroporous adsorption resin column (model LX-28). Stop loading the column when the effluent has a sweet taste. After stopping loading, wash the column with deionized water at 1 BV / h until neutral. Then, perform gradient washing with 2 column volumes of 20-60% ethanol aqueous solution, followed by elution with 70% ethanol aqueous solution. Collect 65.5 L of the eluent, and then wash the column with water until there is no alcohol odor. The column feed flow rate is 0.8 BV / h throughout.

[0059] H: The obtained eluent was then fed into an alkaline anion exchange resin at a rate of 1.5 BV / h, and 79.0 L of effluent was collected.

[0060] I: The collected effluent was concentrated at a temperature of 70℃ and a vacuum degree of <-0.090Mpa until the solid content was 40%, and then spray-dried to obtain mogroside V, 5200g, with a purity of 99.3%. The spray-drying conditions were: inlet air temperature ≤180℃ and outlet air temperature 80-100℃.

[0061] Example 3

[0062] A method for extracting mogroside V includes the following steps:

[0063] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting bacterial cultures were inoculated into fermentation medium at an inoculum size of 8% v / v. The initial pH of the fermentation medium was 7.0, and the initial airflow rate was 0.5 m³ / h. 3 Fermentation was carried out at a concentration of / L, maintaining a pressure of 0.05 MPa, a fermentation temperature of 37℃, and a stirring speed of 290 rpm. During fermentation, the stirring speed was automatically coupled to the dissolved oxygen level. The temperature was automatically controlled by the fermenter. The pH was maintained by adding 50% ammonia water. The dissolved oxygen (DO) value was kept at 28% for the first 24 hours of fermentation. The culture was continued until the OD value of the bacterial culture reached a certain level. 600 The value was 50, and IPTG was added at a final concentration of 0.4% wt to induce fermentation. The mixture was then fermented at a constant temperature of 30℃ for 24 hours to obtain the fermentation broth.

[0064] B: Centrifuge the fermentation broth to collect the cells, and then resuspend the cells in an 80 μM phosphate buffer solution to obtain a resuspended solution. The OD value of the resuspended solution is maintained at 140-160, and the phosphate buffer solution is 4 times the weight of the cells.

[0065] C: The obtained resuspension was homogenized and then centrifuged to obtain enzyme clear solution (enzyme activity of UGT-MS1 was 38 U / ml and enzyme activity of UGT-MS2 was 45 U / ml); the homogenization conditions were 80 MPa, temperature <37℃, and the treatment was repeated twice.

[0066] D: 4300g of mogroside IIE, 1360g of uridine diphosphate glucose, 6.5L of UGT-MS1, 5.0L of UGT-MS2, and 278g of calcium chloride and magnesium chloride (weight ratio 1:1) were added to 15.0L of phosphate buffer solution. The mixture was stirred at 200rpm to obtain a homogeneous solution. An enzymatic reaction was then carried out under stirring to obtain 35.5L of conversion solution containing mogroside V. The concentrations of mogroside IIE, uridine diphosphate glucose, and salt in the mixture were 180 μm, 80 μm, and 90 μm. The pH of the phosphate buffer solution was 8. The enzyme catalytic reaction was carried out at 40℃ for 2 hours. The dosage of UGT-MS1 was 8 U / ml of the mixture, and the dosage of UGT-MS2 was 7 U / ml.

[0067] E: Heat the conversion solution containing mogroside V to 85°C and keep it at this temperature for 35 minutes. Then add calcium chloride (4% of the volume of the conversion solution) and diatomaceous earth (3% of the volume of the conversion solution). Then filter to obtain 38.0 L of filtrate.

[0068] F: The obtained filtrate is passed through an ultrafiltration membrane, and 52.0 L of the ultrafiltration membrane supernatant is collected. The pore size of the ultrafiltration membrane is 10000 Da, and its purpose is to remove large protein molecules.

[0069] G: Then, adjust the pH of the obtained ultrafiltration membrane supernatant to 8.0, and then load it onto a macroporous adsorption resin column (model LX-28). Stop loading the column when the effluent has a sweet taste. After stopping loading, wash the column with deionized water at 1.5 BV / h until neutral. Then, perform gradient washing with 2 column volumes of 20-60% ethanol aqueous solution, followed by elution with 70% ethanol aqueous solution. Collect 70.5 L of the eluent, and then wash the column with water until there is no alcohol odor. The column feed flow rate is 1.0 BV / h throughout.

[0070] H: The obtained eluent was then fed into an alkaline anion exchange resin at a rate of 2 BV / h, and 85.0 L of effluent was collected.

[0071] I: The collected effluent was concentrated at a temperature of 80℃ and a vacuum degree of <-0.090Mpa until the solid content was 50%, and then spray-dried to obtain 6200g of mogroside V with a purity of 99.5%. The spray-drying conditions were: inlet air temperature ≤180℃ and outlet air temperature 80-100℃.

[0072] Example 4

[0073] A method for extracting mogroside V includes the following steps:

[0074] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting bacterial cultures were inoculated into fermentation medium at an inoculum size of 10% v / v. The initial pH of the fermentation medium was 7.0, and the initial airflow rate was 0.5 m³ / h. 3 Fermentation was carried out at a concentration of 0.05 MPa, a pressure of 38°C, and a rotation speed of 300 rpm. The stirring speed was automatically coupled to the dissolved oxygen level during fermentation. The temperature was automatically controlled by the fermenter. The pH was maintained by adding 50% ammonia solution. The dissolved oxygen (DO) value was kept at 30% for the first 24 hours of fermentation. The culture was continued until the OD value of the bacterial culture reached a certain level. 600 The value was 60, and IPTG was added at a final concentration of 0.4% wt to induce fermentation. The mixture was then fermented at a constant temperature of 30℃ for 24 hours to obtain the fermentation broth.

[0075] B: Centrifuge the fermentation broth to collect the cells, and then resuspend the cells in a 100 μM phosphate buffer solution to obtain a resuspended solution. The OD value of the resuspended solution is maintained at 140-160, and the phosphate buffer solution is twice the weight of the cells.

[0076] C: The obtained resuspension was homogenized and then centrifuged to obtain enzyme clear solution (enzyme activity of UGT-MS1 was 36.5 U / ml and enzyme activity of UGT-MS2 was 44.5 U / ml); the homogenization conditions were 100 MPa, temperature <37℃, and the treatment was repeated 4 times.

[0077] D: 4800g of mogroside IIE, 1700g of uridine diphosphate glucose, 8.5L of UGT-MS1, 5.5L of UGT-MS2, and 309g of calcium chloride and magnesium chloride (weight ratio 1:1) were added to 12.0L of phosphate buffer solution. The mixture was stirred at 220rpm to obtain a homogeneous solution. An enzymatic reaction was then carried out under stirring to obtain 36.0L of conversion solution containing mogroside V. The concentrations of mogroside IIE, uridine diphosphate glucose, and salt in the mixture were 200 μM, 100 μM, and 100 μM. The pH of the phosphate buffer solution was 8. The enzyme catalytic reaction was carried out at 45℃ for 1 hour. The dosage of UGT-MS1 was 10 U / ml of the mixture, and the dosage of UGT-MS2 was 8 U / ml.

[0078] E: Heat the conversion solution containing mogroside V to 90°C and keep it at this temperature for 40 minutes. Then add calcium chloride (4% of the volume of the conversion solution) and diatomaceous earth (5% of the volume of the conversion solution). Then filter to obtain 39.5 L of filtrate.

[0079] F: The obtained filtrate is passed through an ultrafiltration membrane, and 52.5 L of the ultrafiltration membrane supernatant is collected. The pore size of the ultrafiltration membrane is 10000 Da, and its purpose is to remove large protein molecules.

[0080] G: Then, adjust the pH of the obtained ultrafiltration membrane supernatant to 7.0, and then load it onto a macroporous adsorption resin column (model LX-28). Stop loading the column when the effluent has a sweet taste. After stopping loading, wash the column with deionized water at 0.5 BV / h until neutral. Then, perform gradient washing with 3 column volumes of 20-60% ethanol aqueous solution, followed by elution with 70% ethanol aqueous solution. Collect 71.0 L of the eluent, and then wash the column with water until there is no alcohol odor. The column feed flow rate is 0.5 BV / h throughout.

[0081] H: The obtained eluent was then fed into an alkaline anion exchange resin at a rate of 2 BV / h, and 91.0 L of effluent was collected.

[0082] I: The collected effluent was concentrated at a temperature of 80℃ and a vacuum degree of <-0.090Mpa until the solid content was 50%, and then spray-dried to obtain 6500g of mogroside V with a purity of 99.0%. The spray-drying conditions were: inlet air temperature ≤180℃ and outlet air temperature 80-100℃.

[0083] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for extracting mogroside V, characterized in that... Includes the following steps: A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured, and the resulting culture broth was then fermented to obtain a fermentation broth. The nucleotide sequence of UGT-MS1 is shown in SEQ ID NO.1, and the nucleotide sequence of UGT-MS2 is shown in SEQ ID NO.

2. B: Centrifuge the fermentation broth to collect the bacterial cells, and then resuspend the bacterial cells in phosphate buffer solution to obtain a resuspended solution; C: The obtained resuspension was homogenized and then centrifuged to obtain the enzyme clear solution; D: Add mogroside IIE, uridine diphosphate glucose, enzyme solution, and salt to phosphate buffer solution, stir at 50-220 rpm to obtain a mixed solution, and carry out enzyme catalytic reaction under stirring to obtain a conversion solution containing mogroside V. E: Heat the conversion solution containing mogroside V to 80-90℃, keep it at this temperature for 20-40 minutes, then add calcium chloride and diatomaceous earth, and then filter to obtain the filtrate; F: Pass the obtained filtrate through an ultrafiltration membrane and collect the supernatant from the ultrafiltration membrane; G: Then adjust the pH of the obtained ultrafiltration membrane supernatant to 7.0-8.0, and then load it onto the column until the column eluent has a sweet taste. After stopping the column loading, wash the column with deionized water until it is neutral, and then elute with 70% ethanol aqueous solution. Collect the eluent and then wash with water until the column has no alcohol smell. H: The obtained eluent is then fed into an alkaline anion exchange resin at a rate of 1-2 BV / h, and the effluent is collected; I: Concentrate the collected effluent to a solid content of 30-50% w / w, and then spray dry to obtain mogroside V.

2. The method for extracting mogroside V as described in claim 1, characterized in that: In step A, the culture medium is inoculated with the bacterial culture at an inoculum size of 5–10% v / v. The initial pH of the fermentation medium is 7.0, the initial air flow rate is 0.5 m³ / L, the maintenance pressure is 0.05 MPa, the fermentation temperature is 35–38°C, and the stirring speed is 250–300 rpm. During fermentation, the stirring speed is automatically coupled to the dissolved oxygen level, the temperature is automatically controlled by the fermenter, and the pH is maintained by adding 50% ammonia water. The dissolved oxygen (DO) value is maintained at 20–30% for the first 24 hours of fermentation. The culture is continued until the OD value of the bacterial culture reaches the settling point. 600 The pH value was 30–60. IPTG was added as an inducer at a final concentration of 0.4% wt, and fermented at a constant temperature of 30°C for 24 hours to obtain the fermentation broth.

3. The method for extracting mogroside V as described in claim 1, characterized in that: In step B, the OD value of the resuspension is maintained at 140-160, the concentration of the phosphate buffer solution is 50-100 μM, and the amount of phosphate buffer solution added is 2-4 times the weight of the bacterial body.

4. The method for extracting mogroside V as described in claim 1, characterized in that: In step C, the homogenizer is operated under the following conditions: 40-100 MPa, temperature <37℃, and the process is repeated 2-4 times.

5. The method for extracting mogroside V as described in claim 1, characterized in that: In step D, the concentration of mogroside IIE in the mixture is 100-200 μM, the concentration of uridine diphosphate glucose is 50-100 μM, the salt is one or more of calcium chloride and magnesium chloride, the concentration of the salt solution is 50-100 μM, and the pH of the phosphate buffer solution is 6-8.

6. The method for extracting mogroside V as described in claim 1, characterized in that: The temperature of the enzyme-catalyzed reaction in step D is 20-45℃, the reaction time is 1-5h, the amount of UGT-MS1 added is 5-10U / ml, and the amount of UGT-MS2 added is 4-8U / ml.

7. The method for extracting mogroside V as described in claim 1, characterized in that: In step E, the amount of calcium chloride added is 2-4% of the volume of the conversion liquid, and the amount of diatomaceous earth added is 3-5% of the volume of the conversion liquid.

8. The method for extracting mogroside V as described in claim 1, characterized in that: In step F, the pore size of the ultrafiltration membrane is 5000-10000 Da.

9. The method for extracting mogroside V as described in claim 1, characterized in that: In step G, the feed rate of the ultrafiltration membrane supernatant into the column is 0.5-1.0 BV / h. The feed flow rate of deionized water is 0.5-1.5 BV / h; After washing the column with deionized water until neutral, perform gradient washing with 1.5-3 column volumes of 20-60% ethanol aqueous solution.

10. The method for extracting mogroside V as described in claim 1, characterized in that: The concentration conditions are a temperature of 60-80℃ and a vacuum degree of <-0.090Mpa; The conditions for spray drying are an inlet air temperature ≤180℃ and an outlet air temperature of 80-100℃.

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