Extraction method of mogroside V

Abstract

The invention discloses an extraction method of mogroside V, and relates to the technical field of biology. Escherichia coli containing glycosyl transferases UGT-MS1 and UGT-MS2 is cultured, fermented, centrifuged and homogenized to obtain enzyme clear liquid, mogroside IIE, uridine diphosphate glucose, the enzyme clear liquid and salt are added into a phosphate buffer solution for enzyme catalysis to obtain conversion liquid, and the conversion liquid is subjected to enzyme purification to obtain the mogroside V. And finally, carrying out ultrafiltration, column loading and elution, then feeding into alkaline anion exchange resin, and concentrating and drying effluent to obtain mogroside V. The product is high in yield and environment-friendly.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting mogroside V. Background Art

[0002] Momordica grosvenorii is the dried mature fruit of the perennial herbaceous vine Momordica grosvenorii of the Cucurbitaceae family. It is also known as Lahan fruit, fairy fruit and light fruit wood turtle, etc. It is one of the first batch of medicine and food homologous drugs in my country. my country has a long history of planting Momordica grosvenorii, which is produced in the cool areas of the south, mainly including Guangxi, Jiangxi and Guizhou. As a traditional Chinese medicine, not only the fruit of Momordica grosvenorii can be used as medicine, but also the roots, leaves and flowers of Momordica grosvenorii have medicinal value. In the theory of traditional Chinese medicine, its functions are mainly to clear the lungs and relieve throat, resolve phlegm and relieve cough, and moisten the intestines and laxatives. Because of its cool nature and the production of body fluid, it has a very obvious effect on the treatment of tracheitis, sore throat, and expectoration. Modern pharmacological activity studies have shown that Momordica grosvenorii also has anti-inflammatory, anti-tumor and antioxidant effects. Although natural products from plants are widely recognized in the current market and have broad application prospects, the research on effective pharmacologically active substances in Momordica grosvenorii is still in its infancy. As a food, monk fruit is rich in vitamins, amino acids and lipids, which have a good promoting effect on the development of the body. Monk fruit is also very sweet and low in calories, making it an excellent sugar substitute.

[0003] Mogroside is the main terpene active substance in monk fruit, belonging to the cucurbitane-type tetracyclic triterpenoid saponin, and is also the main sweet substance in monk fruit, with a sweetness of about 300 times that of sucrose. In mature fruits, mogroside V is the main sweet substance, and it also contains a small amount of mogroside IV. This sweet mogroside has been widely used in the market and is called mogroside; in immature fruits, it is mainly mogroside II and mogroside III. At this time, the fruit has no sweetness and even has a strong bitter taste. In 2010. Monk fruit extract passed the GRAS certification of the Food and Drug Administration (FDA) and was approved as a dietary supplement and natural sweetener. Because mogroside cannot be digested and absorbed by the human body, has a high sweetness, and is a non-sugar natural product, it can be added to cakes, breads and other foods as a low-calorie, high-power sweetener to replace sucrose. It will not cause blood sugar to rise after consumption, which is good news for patients with obesity and diabetes. With the widespread attention of the society, many food and beverage companies have added mogroside to their products in recent years, and the demand for mogroside will increase day by day. In addition, the research on monk fruit extract in traditional Chinese medicine is becoming more and more extensive. Due to the increasing demand for healthy and low-sugar life, mogroside is made into medical throat tablets and non-calorie health products and is widely used.

[0004] At present, the acquisition of mogroside mainly relies on plant extraction methods, but the plant extraction method is mainly affected by the season and the quality of the fruit of the monk fruit. Secondly, the content of mogroside is low due to the different quality of the monk fruit during the extraction process, and the differences between the components are also large, which leads to the need to consume a lot of manpower and material resources in the separation and purification process, and is limited by different separation methods. The most important aspect is that the maturity cycle of the monk fruit is long, and the annual output is far from enough for the growing demand of people. The other two methods for obtaining mogroside are chemical synthesis and enzymatic hydrolysis. The chemical synthesis method is extremely difficult, and multiple steps of chemical protection and deprotection processes are required during the synthesis process. The catalytic efficiency is extremely low, and the demand for energy consumption is very high. In the synthesis process, a variety of chemical reagents are required, which seriously pollutes the environment; the difficulties faced by the enzymatic hydrolysis method are also great. First, the stereoselectivity is relatively poor, and it is impossible to identify specific sites for hydrolysis, and the hydrolysis is difficult to control, and it is impossible to obtain a specific product with high sweetness and good activity. Although the enzymatic hydrolysis method is fast and pollution-free, the yield of mogroside is still very low. Summary of the invention

[0005] The technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a method for extracting mogroside V is provided, which has high yield and is environmentally friendly.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

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

[0008] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 are cultured, and the obtained culture solution is fermented to obtain a fermentation solution, wherein 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: centrifuging the fermentation broth to collect the bacterial cells, and then resuspending the bacterial cells in a phosphate buffer solution to obtain a resuspended liquid;

[0010] C: The obtained resuspension is passed through a homogenizer and then centrifuged to obtain an enzyme clear solution;

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

[0012] E: raising the temperature of the conversion solution containing mogroside V to 80-90° C., keeping the temperature at this temperature for 20-40 minutes, then adding calcium chloride and diatomaceous earth, and filtering to obtain a filtrate;

[0013] F: passing the obtained filtrate through an ultrafiltration membrane and collecting the ultrafiltration membrane clear liquid;

[0014] G: The pH value of the ultrafiltration membrane clear liquid is then adjusted to 7.0-8.0, and then loaded onto the column until the column effluent has a sweet taste. After stopping loading the column, wash the column with deionized water until it is neutral, and then elute it with 70% ethanol aqueous solution, collect the eluate, and then wash with water until the column has no alcohol taste.

[0015] H: The obtained eluate is then passed through a basic anion exchange resin at 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 solution is inoculated into the fermentation medium at an inoculation rate of 5-10% v / v, the initial pH value of the fermentation medium is 7.0, and the initial air flow rate is 0.5 m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 35-38℃, and the rotation speed at 250-300rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 20-30% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 30-60, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

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

[0019] Preferably, the treatment conditions of the homogenizer in step C are 40-100Mpa, temperature <37°C, and treatment is performed 2-4 times.

[0020] Preferably, in step D, the concentration of mogroside IIE is 100-200 Mm; the concentration of UDP-glucose is 50-100 Mm, the salt is one or more of calcium chloride and magnesium chloride, the concentration of the salt is 50-100 Mm, 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 time of the enzyme-catalyzed reaction 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 solution, and the amount of diatomaceous earth added is 3-5% of the volume of the conversion solution.

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

[0024] Preferably, the flow rate of the ultrafiltration membrane clear liquid into the column 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 it is neutral and then gradiently washed with 1.5-3 column volumes and 20-60% ethanol aqueous solution.

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

[0028] The conditions for spray drying are inlet air temperature ≤180℃ and outlet air temperature 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 low in cost, and it reacts with the phosphates in the conversion liquid to form calcium phosphate precipitates, which can aid filtration during the filtration process, speed up the filtration of the filtrate, and achieve the effect of phosphorus removal, reducing the cost of subsequent sewage treatment and being environmentally friendly.

[0035] 2. The obtained filtrate is passed through an ultrafiltration membrane to remove large molecular proteins, which can increase the injection volume and separation efficiency of the subsequent resin.

[0036] 3. By using an anion exchange resin suitable for mogroside V, the color of mogroside V changes from the original light yellow or brownish yellow to a white product, and the eluate of the macroporous resin does not need to be concentrated and recovered by ethanol, but can be directly loaded on the column, thus shortening the production steps and production cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the liquid chromatogram of Mogroside V in Example 3. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments.

[0039] Example 1

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

[0041] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured and the obtained culture solution was inoculated into the fermentation medium at an inoculum volume of 5% v / v. The initial pH value of the fermentation medium was 7.0 and the initial air flow rate was 0.5 m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 35℃, and the rotation speed at 250rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 20% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 30, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

[0042] B: The fermentation broth is centrifuged to collect the bacteria, and then the bacteria are resuspended in a 50 mM phosphate buffer solution to obtain a resuspension. The OD value of the resuspension is maintained at 140-160, wherein the phosphate buffer solution is twice the weight of the bacteria.

[0043] C: The obtained resuspension was passed through a homogenizer and then centrifuged to obtain an enzyme clear solution (the enzyme activity of UGT-MS1 was 35U / ml, and the enzyme activity of UGT-MS2 was 40U / ml); the processing conditions of the homogenizer were 40Mpa, temperature <37°C, and the treatment was repeated 4 times.

[0044] D: 2400g of mogroside IIE, 852g of UDP-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, and stirred evenly at 50rp m to obtain a mixed solution, and an enzyme-catalyzed reaction was carried out under stirring to obtain 31.5L of conversion solution containing mogroside V. The concentration of mogroside IIE in the mixed solution was 100Mm, the concentration of UDP-glucose was 50Mm, the concentration of salt was 50Mm, and the pH of the phosphate buffer solution was 6; the temperature of the enzyme-catalyzed reaction was 20°C, the time of the enzyme-catalyzed reaction was 5h, the amount of UGT-MS1 added was 5U / ml of the mixed solution, and the amount of UGT-MS2 added was 4U / ml of the mixed solution.

[0045] E: The conversion solution containing mogroside V was heated to 80°C and kept at this temperature for 20 min, then calcium chloride (the amount added was 2% of the volume of the conversion solution) and diatomaceous earth (the amount added was 3% of the volume of the conversion solution) were added, and then filtered to obtain 34.0 L of filtrate;

[0046] F: The obtained filtrate is passed through an ultrafiltration membrane, and 49.0 L of ultrafiltration membrane clear liquid is collected. The pore size of the ultrafiltration membrane is 5000 Da, and the purpose is to remove large molecular proteins;

[0047] G: The pH value of the ultrafiltration membrane clear liquid was adjusted to 7.0, and then the macroporous adsorption resin column, model LX-28, was applied until the column effluent had a sweet taste. After the column was stopped, the column was washed with deionized water at 0.5 BV / h until it was neutral, and then gradient washed with 1.5 times the column volume and 20-60% ethanol aqueous solution, and then eluted with 70% ethanol aqueous solution, and 60.0 L of the eluate was collected, and then washed with water until the column had no alcohol taste, wherein the column inlet flow rate was 0.5 BV / h;

[0048] H: The obtained eluate is then fed into a basic anion exchange resin at 1 BV / h, and 75.0 L of the effluent is collected;

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

[0050] Example 2

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

[0052] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured and the obtained culture solution was inoculated into the fermentation medium at an inoculum volume of 6% v / v. The initial pH value of the fermentation medium was 7.0 and the initial air flow rate was 0.5 m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 36℃, and the rotation speed at 270rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 25% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 40, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

[0053] B: The fermentation broth was centrifuged to collect the bacterial cells, and then the bacterial cells were resuspended in a 60 mM phosphate buffer solution to obtain a resuspension. The OD value of the resuspension was maintained at 140-160, wherein the phosphate buffer solution was 3 times the weight of the bacterial cells.

[0054] C: The obtained resuspension was passed through a homogenizer and then centrifuged to obtain an enzyme clear solution ((the enzyme activity of UGT-MS1 is 36U / ml, and the enzyme activity of UGT-MS2 is 42U / ml)); the processing conditions of the homogenizer were 60Mpa, temperature <37°C, and the treatment was repeated 3 times.

[0055] D: 3600g of mogroside IIE, 1192g of UDP-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, and stirred evenly at 100rpm to obtain a mixed solution, and an enzyme-catalyzed reaction was carried out under stirring to obtain 32.0L of conversion solution containing mogroside V. The concentration of mogroside IIE in the mixed solution was 150Mm, the concentration of UDP-glucose was 70Mm, the concentration of salt was 70Mm, and the pH of the phosphate buffer solution was 7; the temperature of the enzyme-catalyzed reaction was 30°C, the time of the enzyme-catalyzed reaction was 3h, the amount of UGT-MS1 added was 8U / ml of the mixed solution, and the amount of UGT-MS2 added was 6U / ml of the mixed solution.

[0056] E: The conversion solution containing mogroside V was heated to 85°C and kept at this temperature for 30 min, then calcium chloride (the amount added was 3% of the volume of the conversion solution) and diatomaceous earth (the amount added was 4% of the volume of the conversion solution) were added, and then filtered to obtain 34.5 L of filtrate;

[0057] F: The obtained filtrate is passed through an ultrafiltration membrane, and 50.5 L of ultrafiltration membrane clear liquid is collected. The pore size of the ultrafiltration membrane is 8000 Da, and the purpose is to remove large molecular proteins;

[0058] G: The pH value of the ultrafiltration membrane clear liquid was adjusted to 8.0, and then the macroporous adsorption resin column, model LX-28, was applied until the column effluent had a sweet taste, and then the column was washed with deionized water at 1BV / h until it was neutral, and then gradient washed with 2 times the column volume and 20-60% ethanol aqueous solution, and then eluted with 70% ethanol aqueous solution, and the eluate was collected. 65.5L, and then washed with water until the column had no alcohol taste. The column flow rate was 0.8BV / h;

[0059] H: The obtained eluate is then fed into a basic anion exchange resin at 1.5 BV / h, and 79.0 L of the effluent is collected;

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

[0061] Example 3

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

[0063] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured and the obtained culture solution was inoculated into the fermentation medium at an inoculum volume of 8% v / v. The initial pH value of the fermentation medium was 7.0 and the initial air flow rate was 0.5 m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 37°C, and the rotation speed at 290rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 28% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 50, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

[0064] B: The fermentation broth was centrifuged to collect the bacterial cells, and then the bacterial cells were resuspended in a phosphate buffer solution with a concentration of 80 mM to obtain a resuspension. The OD value of the resuspension was maintained at 140-160, wherein the phosphate buffer solution was 4 times the weight of the bacterial cells.

[0065] C: The obtained resuspension was passed through a homogenizer and then centrifuged to obtain an enzyme clear solution (the enzyme activity of UGT-MS1 was 38U / ml, and the enzyme activity of UGT-MS2 was 45U / ml); the processing conditions of the homogenizer were 80Mpa, temperature <37°C, and the treatment was repeated twice.

[0066] D: 4300g of mogroside IIE, 1360g of UDP-glucose, 6.5L of UGT-MS1, 5.0L of UGT-MS2, 278g of calcium chloride and magnesium chloride (weight ratio 1:1) were added to 15.0L of phosphate buffer solution, stirred evenly at 200rpm to obtain a mixed solution, and an enzyme-catalyzed reaction was carried out under stirring to obtain 35.5L of conversion solution containing mogroside V. The concentration of mogroside IIE in the mixed solution was 180Mm, the concentration of UDP-glucose was 80Mm, the concentration of salt was 90Mm, the pH of the phosphate buffer solution was 8; the temperature of the enzyme-catalyzed reaction was 40°C, the time of the enzyme-catalyzed reaction was 2h, the amount of UGT-MS1 added was 8U / ml of the mixed solution, and the amount of UGT-MS2 added was 7U / ml of the mixed solution.

[0067] E: The conversion solution containing mogroside V was heated to 85°C, kept at this temperature for 35 minutes, and then calcium chloride (4% of the volume of the conversion solution) and diatomaceous earth (3% of the volume of the conversion solution) were added, and then filtered to obtain 38.0 L of filtrate;

[0068] F: The obtained filtrate is passed through an ultrafiltration membrane, and 52.0 L of ultrafiltration membrane clear liquid is collected. The pore size of the ultrafiltration membrane is 10000 Da, and the purpose is to remove large molecular proteins;

[0069] G: The pH value of the ultrafiltration membrane clear liquid was adjusted to 8.0, and then the macroporous adsorption resin column, model LX-28, was applied until the column effluent had a sweet taste, and then the column was washed with deionized water at 1.5BV / h until it was neutral, and then gradient washed with 2 times the column volume and 20-60% ethanol aqueous solution, and then eluted with 70% ethanol aqueous solution, and the eluate was collected. 70.5L, and then washed with water until the column had no alcohol taste. The column flow rate was 1.0BV / h;

[0070] H: The obtained eluate is then fed into a basic anion exchange resin at 2 BV / h, and 85.0 L of the effluent is collected;

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

[0072] Example 4

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

[0074] A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 were cultured and the obtained culture solution was inoculated into the fermentation medium at an inoculum of 10% v / v. The initial pH value of the fermentation medium was 7.0 and the initial air flow rate was 0.5 m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 38℃, and the rotation speed at 300rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 30% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 60, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

[0075] B: The fermentation broth was centrifuged to collect the bacterial cells, and then the bacterial cells were resuspended in a 100 mM phosphate buffer solution to obtain a resuspension. The OD value of the resuspension was maintained at 140-160, wherein the phosphate buffer solution was twice the weight of the bacterial cells.

[0076] C: The obtained resuspension was passed through a homogenizer and then centrifuged to obtain an enzyme clear solution (the enzyme activity of UGT-MS1 was 36.5U / ml, and the enzyme activity of UGT-MS2 was 44.5U / ml); the processing conditions of the homogenizer were 100Mpa, temperature <37°C, and the treatment was repeated 4 times.

[0077] D: 4800g of mogroside IIE, 1700g of UDP-glucose, 8.5L of UGT-MS1, 5.5L of UGT-MS2, 309g of calcium chloride and magnesium chloride (weight ratio 1:1) were added to 12.0L of phosphate buffer solution, stirred evenly at 220rpm to obtain a mixed solution, and an enzyme-catalyzed reaction was carried out under stirring to obtain 36.0L of conversion solution containing mogroside V. The concentration of mogroside IIE in the mixed solution was 200Mm, the concentration of UDP-glucose was 100Mm, the concentration of salt was 100Mm, the pH of the phosphate buffer solution was 8; the temperature of the enzyme-catalyzed reaction was 45°C, the time of the enzyme-catalyzed reaction was 1h, the amount of UGT-MS1 added was 10U / ml of the mixed solution, and the amount of UGT-MS2 added was 8U / ml of the mixed solution.

[0078] E: The conversion solution containing mogroside V was heated to 90°C and kept at this temperature for 40 minutes, then calcium chloride (4% of the volume of the conversion solution) and diatomaceous earth (5% of the volume of the conversion solution) were added, and then filtered to obtain 39.5 L of filtrate;

[0079] F: The obtained filtrate is passed through an ultrafiltration membrane, and 52.5 L of ultrafiltration membrane clear liquid is collected. The pore size of the ultrafiltration membrane is 10000 Da, and the purpose is to remove large molecular proteins;

[0080] G: The pH value of the ultrafiltration membrane clear liquid was adjusted to 7.0, and then the macroporous adsorption resin column, model LX-28, was applied until the column effluent had a sweet taste, and then the column was washed with deionized water at 0.5BV / h until it was neutral, and then gradient washed with 3 times column volume and 20-60% concentration of ethanol aqueous solution, and then eluted with 70% ethanol aqueous solution, and the eluate was collected. 71.0L, and then washed with water until the column had no alcohol taste. The column flow rate was 0.5BV / h;

[0081] H: The obtained eluate is then fed into a basic anion exchange resin at 2 BV / h, and 91.0 L of the effluent is collected;

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

[0083] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for extracting mogroside V, characterized in that The following steps are involved: A: Escherichia coli containing glycosyltransferases UGT-MS1 and UGT-MS2 are cultured, and the obtained culture solution is fermented to obtain a fermentation solution, wherein 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: centrifuging the fermentation broth to collect the bacterial cells, and then resuspending the bacterial cells in a phosphate buffer solution to obtain a resuspended liquid; C: The obtained resuspension is passed through a homogenizer and then centrifuged to obtain an enzyme clear solution; D: Add mogroside IIE, uridine diphosphate glucose, enzyme supernatant and salt into phosphate buffer solution, stir evenly at 50-220 rpm to obtain a mixed solution, and carry out enzyme catalysis reaction under stirring to obtain a conversion solution containing mogroside V. E: raising the temperature of the conversion solution containing mogroside V to 80-90° C., keeping the temperature at this temperature for 20-40 minutes, then adding calcium chloride and diatomaceous earth, and filtering to obtain a filtrate; F: passing the obtained filtrate through an ultrafiltration membrane and collecting the ultrafiltration membrane clear liquid; G: The pH value of the ultrafiltration membrane clear liquid is then adjusted to 7.0-8.0, and then loaded onto the column until the column effluent has a sweet taste. After stopping loading the column, wash the column with deionized water until it is neutral, and then elute it with 70% ethanol aqueous solution, collect the eluate, and then wash with water until the column has no alcohol taste. H: The obtained eluate is then passed through a basic anion exchange resin at 1-2 BV / h, and the effluent is collected; I: The collected effluent is concentrated to a solid content of 30-50% w / w, and then spray-dried to obtain mogroside V.

2. A method according to claim 1, characterized in that: In step A, the culture solution is inoculated into the fermentation medium at an inoculum rate of 5-10% v / v, the initial pH value of the fermentation medium is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.05Mpa, the fermentation temperature at 35-38℃, and the rotation speed at 250-300rpm. During the fermentation process, the stirring speed is automatically coupled and set according to the dissolved oxygen content. The temperature is automatically controlled by the fermentation tank. The pH is maintained by adding 50% ammonia water by volume. The DO value is maintained at 20-30% before fermentation for 24 hours. The culture is cultured until the bacterial solution OD 600 The value was 30-60, and the inducer IPTG was added with a final concentration of 0.4% wt for induction, and the mixture was fermented at a constant temperature of 30°C for 24 hours to obtain a fermentation broth.

3. A method according to 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 Mm, and the amount of phosphate buffer solution added is 2-4 times the weight of the bacterial cell.

4. A method as claimed in claim 1, characterized in that: The processing conditions of the homogenizer in step C are 40-100Mpa, temperature <37°C, and the processing is 2-4 times.

5. A method according to claim 1, characterized in that: In step D, the concentration of mogroside IIE in the mixed solution is 100-200 Mm, the concentration of UDP-glucose is 50-100 Mm, the salt is one or more of calcium chloride and magnesium chloride, the concentration of the salt solution is 50-100 Mm, and the pH of the phosphate buffer solution is 6-8.

6. A method according to claim 1, characterized in that: The temperature of the enzyme catalyzed reaction in step D is 20-45° C., the time of the enzyme catalyzed reaction 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.

7. A method according to claim 1, characterized in that: In step E, the amount of calcium chloride added is 2-4% of the volume of the conversion solution, and the amount of diatomaceous earth added is 3-5% of the volume of the conversion solution.

8. A method according to claim 1, characterized in that: The pore size of the ultrafiltration membrane in step F is 5000-10000Da.

9. A method according to claim 1, characterized in that: In step G, the flow rate of the ultrafiltration membrane clear liquid into the column is 0.5-1.0 BV / h; The feed flow rate of deionized water is 0.5-1.5 BV / h; The column was washed with deionized water until neutral and then gradiently washed with 1.5-3 column volumes and 20-60% ethanol aqueous solution.

10. A method according to claim 1, characterized in that: The concentration conditions are temperature 60-80°C and vacuum degree <-0.090Mpa; The conditions for spray drying are inlet air temperature ≤180℃ and outlet air temperature 80-100℃.

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