A process for producing nicotinamide riboside by high-density fermentation

Abstract

The present invention belongs to the field of biotechnology, and provides a process for producing nicotinamide riboside by high-density fermentation, comprising the following steps: step 1, preparing culture medium and solution: preparing LB culture medium, fermentation culture medium, nicotinamide solution and glucose solution; step 2, preparing seed solution; step 3, fermentation; step 4, filtering the fermentation broth obtained in step 3 once and twice in sequence, wherein the first filtration is filtered by ultrafiltration, and the second filtration is filtered by microfiltration to obtain a filtrate; step 5, adding the filtrate obtained in step 4 and water to a reactor, and then adding 3-cyanopyridine to react to obtain a hydrolyzate; step 6, ultrafiltration is used to ultrafilter the hydrolyzate obtained in step 5, followed by reduced pressure concentration, and finally spray drying and granulation to obtain nicotinamide riboside. A process for producing nicotinamide riboside by high-density fermentation provided by the present invention has the advantages of high reaction yield and mild conditions.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a process for producing nicotinamide riboside by high-density fermentation. Background Art

[0002] Nicotinamide riboside (NR) is a derivative of vitamin B3. It can react with phosphate and adenine to form nicotinamide adenine dinucleotide (NAD, also known as coenzyme I) and nicotinamide adenine dinucleotide phosphate (NADP, also known as coenzyme II). It is an important coenzyme NAD. + Nicotinamide riboside plays an important role in the energy generation of human cells. It participates in the synthesis of intracellular nicotinamide adenine dinucleotide (an important coenzyme for cellular energy conversion). Nicotinamide adenine dinucleotide is a coenzyme that transfers protons. It appears in many metabolic reactions of cells and participates in the decomposition of compounds such as proteins, carbohydrates and fats. For example, chiral reduction catalyzed by leucine dehydrogenase, formate ammonia dehydrogenase and glucose dehydrogenase requires the help of nicotinamide adenine dinucleotide to complete the entire reaction, and the same is true for oxidation reactions. As cells age or become diseased, the amount of nicotinamide adenine dinucleotide decreases. Therefore, supplementing nicotinamide riboside and increasing the content of nicotinamide adenine dinucleotide can improve the basic metabolic activities of cells, thereby significantly improving cell vitality and improving various aspects of human physiological functions.

[0003] At present, there are many methods for preparing nicotinamide: the traditional reaction technology is chemical hydrolysis, using alkaline substances as catalysts, and various hydrolysis processes produced by different catalysts and different recovery and separation methods. However, the conversion reaction of this chemical hydrolysis process is relatively single, and the conversion rate and reaction conditions are in a contradictory relationship. When the pH value increases, the conversion rate increases, but the output of the side reaction product nicotinic acid also increases. On the contrary, when the pH value is reduced to reduce the output of the side reaction product, the reaction product decreases again, and excessively reducing the pH value will lead to a decrease in the quality of nicotinamide, and some preparation methods will produce byproducts that corrode the equipment, and there are safety problems. The catalyst is expensive, the energy consumption is large, and the income is unstable, resulting in high costs. In the Chinese invention application patent with publication number CN110452277A, nicotinamide and 1,2,3,5-tetraacetyl-β-D-furanose are used as substrates, and the finished product NR is obtained through condensation reaction, acetyl deprotection reaction, column chromatography, distillation drying, and recrystallization. In the Chinese invention patent application with publication number CN111763235A, NR is synthesized by using furanose, methyl nicotinate, and chloroform, and adding trimethylsilyl trifluoromethanesulfonate as a catalyst. The conversion efficiency of chemical synthesis is relatively high, but the addition of catalysts and toxic reagents limits the application of the generated NR in the food field.

[0004] Therefore, how to develop a production process for preparing nicotinamide riboside with high yield and high efficiency is a technical problem that urgently needs to be solved. Summary of the invention

[0005] In order to increase the yield of nicotinamide riboside, the present invention provides a process for producing nicotinamide riboside by high-density fermentation.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A process for producing nicotinamide riboside by high-density fermentation comprises the following steps:

[0008] Step 1, preparing culture medium and solution: preparing LB culture medium, fermentation medium, nicotinamide solution and glucose solution;

[0009] Step 2, prepare seed solution: Sterilize LB solid medium and spread it on the double-resistance plate for cultivation, pick out single bacteria from the double-resistance plate and streak it on a new double-resistance plate for further cultivation, streak the strain and inoculate it on LB medium, add Kan and Sm and continue to cultivate, OD 600 When the pH value is 1.5-2.5, the culture is terminated and the seed solution is obtained;

[0010] Step 3, fermentation: inoculate the seed liquid into the fermentation tank, then add the fermentation medium, adjust the pH, maintain the temperature at 30-37°C, and the dissolved oxygen (DO) at 10-30%; after the DO rises and the glucose in the medium is consumed, add the glucose solution to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600 ≥15-20, add IPTG for induction, add nicotinamide solution after induction, maintain the content of nicotinamide ≤0.5g / L during the fermentation process, and obtain fermentation liquid;

[0011] Step 4, filtering the fermentation broth obtained in step 3 once and twice in sequence to obtain a filtrate;

[0012] Step 5, adding the filtrate obtained in step 4 and water into a reaction kettle, the reaction temperature is 23-25° C., and then adding 3-cyanopyridine to react to obtain a hydrolyzate;

[0013] Step 6: The hydrolyzate obtained in step 5 is ultrafiltered by ultrafiltration, then concentrated under reduced pressure, spray dried, and granulated to obtain nicotinamide riboside.

[0014] As a further solution of the present invention, the LB medium in step 1 contains 5-6 g / L yeast extract powder, 8-10 g / L peptone, 10-11 g / L sodium chloride, and 15-18 g / L agar strips are added to prepare LB solid medium.

[0015] As a further embodiment of the present invention, the fermentation medium in step 1 contains KH2PO4 6.67-6.70 g / L, citric acid 1.67-1.70 g / L, ammonium sulfate 1.50-1.55 g / L, glucose 10-20 g / L, MgSO4·7H2O 1.5-5 g / L, yeast powder 0.2-1 g / L, ferrous sulfate 0.05-0.1 g / L, manganese sulfate 0.05-0.1 g / L, CoCl2 0.001-0.002 g / L, VB1 0.01-0.02 g / L, VH 0.01-0.02 g / L, defoamer 0.01%, Kan 0.02 g / L, and ST 0.02 g / L.

[0016] As a further solution of the present invention, the nicotinamide solution in step 1 is prepared by the following steps:

[0017] Dissolve nicotinamide in pure water, place in a sterilizer, and sterilize at 121°C for 20 minutes; the dosage ratio of nicotinamide to pure water is 1g:10mL.

[0018] As a further solution of the present invention, the glucose solution in step 1 is prepared by the following steps:

[0019] Dissolve glucose monohydrate in pure water, place in an autoclave, and sterilize at 115°C for 20 minutes; the dosage ratio of glucose monohydrate to pure water is 6g:10mL.

[0020] As a further embodiment of the present invention, the final concentration of Kan and Sm in step 2 is 50 mg / L.

[0021] As a further solution of the present invention, in step 3, the pH is adjusted to 6.7-7.2; the concentration of IPTG is 0.1-0.5 mM / L.

[0022] As a further solution of the present invention, in step 4, the first filtration is performed by ultrafiltration, and the second filtration is performed by microfiltration; wherein the pore size of the ultrafiltration membrane is The pore size of the microfiltration membrane is 1-3 μm.

[0023] As a further solution of the present invention, in step 5, the volume ratio of the filtrate to water is 1:(5-7); the volume ratio of the filtrate to 3-cyanopyridine is 1:2.

[0024] As a further solution of the present invention, the temperature during the reduced pressure concentration is 68-75°C, the pressure is 18-25KPa, and the time is 5-7h.

[0025] Beneficial effects of the present invention:

[0026] The process for producing nicotinamide riboside by high-density fermentation provided by the present invention is to select biological strains for cultivation, and then adjust and optimize the fermentation conditions, such as temperature, oxygen concentration and substrate concentration, to achieve high-density fermentation of biological strains, thereby efficiently producing nicotinamide riboside. The method provided by the present invention has the advantages of mild reaction conditions and high yield.

[0027] The present invention adopts high-density fermentation production to prepare nicotinamide riboside. Compared with the traditional chemical synthesis method, the biological method not only reduces environmental pollution, but also reduces production costs. At the same time, the cost of raw materials and solvents used is low, and the economy is good. At the same time, the reaction conditions are mild, and the product separation is easy. The obtained nicotinamide riboside has a high yield, high purity, and low pollution, which conforms to the concept of green production and is suitable for large-scale production.

[0028] The process for producing nicotinamide riboside by fermentation provided in the present invention can simplify equipment investment and further reduce production costs because the fermentation method has relatively low requirements on equipment. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The strains used in the examples and comparative examples of the present invention are all Escherichia coli.

[0031] Example 1

[0032] This embodiment provides a process for producing nicotinamide riboside by high-density fermentation, comprising the following steps:

[0033] Step 1: Prepare culture medium and solution:

[0034] LB medium: 5 g / L yeast extract powder, 8 g / L peptone, 10 g / L sodium chloride, add 15 g / L agar strip to prepare LB solid medium;

[0035] Fermentation medium: KH2PO46.67 g / L, citric acid1.67 g / L, ammonium sulfate1.50 g / L, glucose10 g / L, MgSO4·7H2O1.5 g / L, yeast powder0.2 g / L, ferrous sulfate0.05 g / L, manganese sulfate0.05 g / L, CoCl20.001 g / L, VB10.01 g / L, VH 0.01 g / L, defoamer0.01%, Kan0.02 g / L, ST 0.02 g / L;

[0036] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0037] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min;

[0038] Step 2: Prepare seed solution:

[0039] After sterilizing the LB solid medium at 121°C for 20 min, the strain was spread on the double-resistance plate and cultured at 37°C for 12 h. Then, 30 single bacteria from the double-resistance plate were picked and streaked on a new double-resistance plate. The plates were cultured at 37°C for 12 h. The strains were streaked and inoculated into the LB medium. Kan and Sm with a final concentration of 50 mg / L were added and cultured at 37°C for 10 h. The OD 600 When the pH value is 1.5-2.5, the culture is terminated and the seed solution is obtained;

[0040] Step 3: Fermentation:

[0041] The seed liquid is inoculated into the fermentation tank, and then the fermentation medium is added. During the fermentation process, ammonia water is used to adjust the pH to 6.7, the temperature is maintained at 37°C, and the dissolved oxygen (DO) is 10-30%; after the DO rises and the glucose in the medium is consumed, the glucose solution is added to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600 ≥15-20, control the temperature to 30°C, add 0.1mM / L IPTG for induction, add nicotinamide solution after induction, initially add 0.5g / (L·h) nicotinamide solution, maintain the content of nicotinamide ≤0.5g / L during the fermentation process, and obtain fermentation liquid;

[0042] Step 4: The fermentation liquid obtained in step 3 is filtered once and twice in sequence, wherein the first filtration is performed by ultrafiltration, and the pore size of the ultrafiltration membrane is The secondary filtration was carried out by microfiltration, the pore size of the microfiltration membrane was 1 μm, and the filtrate was obtained;

[0043] Step 5, adding the filtrate obtained in step 4 and water into a reactor in a volume ratio of 1:5, the reaction temperature is 25°C, and then adding 3-cyanopyridine to react in an amount of 2 times the volume of the filtrate to obtain a hydrolyzate;

[0044] Step 6: The hydrolyzate obtained in step 5 is ultrafiltered by ultrafiltration, and then concentrated under reduced pressure at a temperature of 68° C., a pressure of 18 KPa, and a time of 5 h. Finally, it is spray-dried and granulated to obtain nicotinamide riboside.

[0045] Example 2

[0046] This embodiment provides a process for producing nicotinamide riboside by high-density fermentation, comprising the following steps:

[0047] Step 1: Prepare culture medium and solution:

[0048] LB medium: 6 g / L yeast extract powder, 10 g / L peptone, 11 g / L sodium chloride, add 18 g / L agar strip to prepare LB solid medium;

[0049] Fermentation medium: KH2PO46.67 g / L, citric acid1.67 g / L, ammonium sulfate1.50 g / L, glucose10 g / L, MgSO4·7H2O1.5 g / L, yeast powder0.2 g / L, ferrous sulfate0.05 g / L, manganese sulfate0.05 g / L, CoCl20.001 g / L, VB10.01 g / L, VH 0.01 g / L, defoamer0.01%, Kan0.02 g / L, ST 0.02 g / L;

[0050] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0051] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0052] Step 2: Prepare seed solution:

[0053] After sterilizing the LB solid medium at 121°C for 20 min, the strain was spread on the double-resistance plate and cultured at 37°C for 12 h. Then, 30 single bacteria from the double-resistance plate were picked and streaked on a new double-resistance plate. The plates were cultured at 37°C for 12 h. The strains were streaked and inoculated into the LB medium. Kan and Sm with a final concentration of 50 mg / L were added and cultured at 37°C for 10 h. The OD 600 When the pH value is 1.5-2.5, the culture is terminated and the seed solution is obtained;

[0054] Step 3: Fermentation:

[0055] The seed liquid is inoculated into the fermentation tank, and then the fermentation medium is added. During the fermentation process, ammonia water is used to adjust the pH to 6.7, the temperature is maintained at 37°C, and the dissolved oxygen (DO) is 10-30%; after the DO rises and the glucose in the medium is consumed, the glucose solution is added to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600≥15-20, control the temperature to 30°C, add 0.1mM / L IPTG for induction, add nicotinamide solution after induction, initially add 0.5g / (L·h) nicotinamide solution, maintain the content of nicotinamide ≤0.5g / L during the fermentation process, and obtain fermentation liquid;

[0056] Step 4: The fermentation liquid obtained in step 3 is filtered once and twice in sequence, wherein the first filtration is performed by ultrafiltration, and the pore size of the ultrafiltration membrane is The secondary filtration was carried out by microfiltration, the pore size of the microfiltration membrane was 1 μm, and the filtrate was obtained;

[0057] Step 5, adding the filtrate obtained in step 4 and water into a reactor in a volume ratio of 1:5, the reaction temperature is 25°C, and then adding 3-cyanopyridine to react in an amount of 2 times the volume of the filtrate to obtain a hydrolyzate;

[0058] Step 6: The hydrolyzate obtained in step 5 is ultrafiltered by ultrafiltration, and then concentrated under reduced pressure at a temperature of 68° C., a pressure of 18 KPa, and a time of 5 h. Finally, it is spray-dried and granulated to obtain nicotinamide riboside.

[0059] Example 3

[0060] The only difference from Example 1 is that:

[0061] Step 1: Prepare culture medium and solution:

[0062] LB medium: 5.5 g / L yeast extract powder, 9 g / L peptone, 10 g / L sodium chloride, add 16 g / L agar strip to prepare LB solid medium;

[0063] Fermentation medium: KH2PO46.70 g / L, citric acid1.70 g / L, ammonium sulfate1.53 g / L, glucose15 g / L, MgSO4·7H2O2.0 g / L, yeast powder0.8 g / L, ferrous sulfate0.05 g / L, manganese sulfate0.05 g / L, CoCl20.001 g / L, VB10.01 g / L, VH0.01 g / L, defoamer0.01%, Kan0.02 g / L, ST0.02 g / L;

[0064] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0065] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0066] Example 4

[0067] The only difference from Example 1 is that:

[0068] Step 1: Prepare culture medium and solution:

[0069] LB medium: 6 g / L yeast extract powder, 8 g / L peptone, 11 g / L sodium chloride, add 17 g / L agar strip to prepare LB solid medium;

[0070] Fermentation medium: KH2PO46.70 g / L, citric acid 1.70 g / L, ammonium sulfate 1.52 g / L, glucose 12 g / L, MgSO4·7H2O 1.5 g / L, yeast powder 0.2 g / L, ferrous sulfate 0.1 g / L, manganese sulfate 0.1 g / L, CoCl2 0.002 g / L, VB1 0.01 g / L, VH 0.01 g / L, defoamer 0.01%, Kan 0.02 g / L, ST 0.02 g / L;

[0071] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0072] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0073] Example 5

[0074] The only difference from Example 1 is that:

[0075] Step 3: Fermentation:

[0076] The seed liquid is inoculated into the fermentation tank, and then the fermentation medium is added. During the fermentation process, ammonia water is used to adjust the pH to 7.2, the temperature is maintained at 37°C, and the dissolved oxygen (DO) is 10-30%; after the DO rises and the glucose in the medium is consumed, the glucose solution is added to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600 ≥15-20, control the temperature to 30°C, add 0.5mM / L IPTG for induction, add nicotinamide solution after induction, initially add 0.5g / (L·h) nicotinamide solution, maintain the content of nicotinamide ≤0.5g / L during the fermentation process, and obtain fermentation liquid.

[0077] Example 6

[0078] The only difference from Example 1 is that:

[0079] Step 4: The fermentation liquid obtained in step 3 is filtered once and twice in sequence, wherein the first filtration is performed by ultrafiltration, and the pore size of the ultrafiltration membrane is The secondary filtration is carried out by microfiltration, the pore size of the microfiltration membrane is 3 μm, and a filtrate is obtained.

[0080] Example 7

[0081] The only difference from Example 1 is that:

[0082] Step 5: Add the filtrate obtained in step 4 and water into the reactor at a volume ratio of 1:7, the reaction temperature is 23-25° C., and then add 3-cyanopyridine to react in an amount twice the volume of the filtrate to obtain a hydrolyzate.

[0083] Example 8

[0084] The only difference from Example 1 is that:

[0085] Step 6: The hydrolyzate obtained in step 5 is ultrafiltered by ultrafiltration, and then concentrated under reduced pressure at a temperature of 68° C., a pressure of 25 KPa, and a time of 7 h. Finally, it is spray-dried and granulated to obtain nicotinamide riboside.

[0086] Comparative Example 1

[0087] The only difference from Example 1 is that:

[0088] Step 1: Prepare culture medium and solution:

[0089] LB medium: 3 g / L yeast extract powder, 8 g / L peptone, 10 g / L sodium chloride, add 15 g / L agar strip to prepare LB solid medium;

[0090] Fermentation medium: KH2PO46.67 g / L, citric acid1.67 g / L, ammonium sulfate1.50 g / L, glucose10 g / L, MgSO4·7H2O1.5 g / L, yeast powder0.2 g / L, ferrous sulfate0.05 g / L, manganese sulfate0.05 g / L, CoCl20.001 g / L, VB10.01 g / L, VH 0.01 g / L, defoamer0.01%, Kan0.02 g / L, ST 0.02 g / L;

[0091] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0092] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0093] Comparative Example 2

[0094] The only difference from Example 1 is that:

[0095] Step 1: Prepare culture medium and solution:

[0096] LB medium: 5 g / L yeast extract powder, 8 g / L peptone, 10 g / L sodium chloride, add 15 g / L agar strip to prepare LB solid medium;

[0097] Fermentation medium: KH2PO45.67 g / L, citric acid1.67g / L, ammonium sulfate1.50g / L, glucose10g / L, MgSO4·7H2O1.5g / L, yeast powder0.2g / L, ferrous sulfate0.05g / L, manganese sulfate0.05g / L, CoCl20.001 g / L, VB10.01 g / L, VH 0.01g / L, defoamer0.01%, Kan0.02g / L, ST 0.02g / L;

[0098] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0099] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0100] Comparative Example 3

[0101] The only difference from Example 1 is that:

[0102] Step 1: Prepare culture medium and solution:

[0103] LB medium: 4.5 g / L yeast extract powder, 12 g / L peptone, 10 g / L sodium chloride, add 15 g / L agar strip to prepare LB solid medium;

[0104] Fermentation medium: KH2PO46.67 g / L, citric acid1.67 g / L, ammonium sulfate1.50 g / L, glucose10 g / L, MgSO4·7H2O1.5 g / L, yeast powder0.2 g / L, ferrous sulfate0.05 g / L, manganese sulfate0.05 g / L, CoCl20.001 g / L, VB10.01 g / L, VH 0.01 g / L, defoamer0.01%, Kan0.02 g / L, ST 0.02 g / L;

[0105] Nicotinamide solution: Dissolve 100g nicotinamide in 1000mL pure water, place in a sterilizer, and sterilize at 121℃ for 20min;

[0106] Glucose solution: Dissolve 600 g of glucose monohydrate in 1000 mL of pure water, place in an autoclave, and sterilize at 115°C for 20 min.

[0107] Comparative Example 4

[0108] The only difference from Example 1 is that:

[0109] Step 2: Prepare seed solution:

[0110] After sterilizing the LB solid medium at 121°C for 20 min, 40 μL Kan and 40 μL Sm were added, and then the strain was spread on the double-resistance plate. After culturing at 37°C for 12 h, 30 single bacteria from the double-resistance plate were picked and streaked on a new double-resistance plate. The plates were cultured at 37°C for 12 h, and the strains were inoculated into the LB medium. Kan and Sm with a final concentration of 50 mg / L were added, and the plates were cultured at 37°C for 8 h. The OD 600 When the value is 1, the culture is terminated and the seed solution is obtained.

[0111] Comparative Example 5

[0112] The only difference from Example 1 is that:

[0113] Step 3: Fermentation:

[0114] The seed liquid is inoculated into the fermentation tank, and then the fermentation medium is added. During the fermentation process, ammonia water is used to adjust the pH to 5.5, the temperature is maintained at 37°C, and the dissolved oxygen (DO) is 10-30%; after the DO rises and the glucose in the medium is consumed, the glucose solution is added to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600 ≥15-20, control the temperature at 28-30°C, add 0.1mM / L IPTG for induction, add nicotinamide solution after induction, initially add 0.5g / (L·h) nicotinamide solution, maintain the nicotinamide content ≤0.5g / L during the fermentation process, and obtain fermentation liquid.

[0115] Comparative Example 6

[0116] The only difference from Example 1 is that:

[0117] Step 4: The fermentation liquid obtained in step 3 is filtered once and twice in sequence, wherein the first filtration is performed by ultrafiltration, and the pore size of the ultrafiltration membrane is The secondary filtration is carried out by microfiltration, the pore size of the microfiltration membrane is 0.5 μm, and a filtrate is obtained.

[0118] Comparative Example 7

[0119] The only difference from Example 1 is that:

[0120] Step 5: Add the filtrate obtained in step 4 and water into the reactor at a volume ratio of 1:3, the reaction temperature is 25° C., and then add 3-cyanopyridine to react in an amount twice the volume of the filtrate to obtain a hydrolyzate.

[0121] Comparative Example 8

[0122] The only difference from Example 1 is that:

[0123] Step 6: The hydrolyzate obtained in step 5 is ultrafiltered by ultrafiltration, and then concentrated under reduced pressure at a temperature of 75° C., a pressure of 15 KPa, and a time of 7 h. Finally, it is spray-dried and granulated to obtain nicotinamide riboside.

[0124] The following tests were performed on Examples 1-8 and Comparative Examples 1-8:

[0125] Using a chromatographic column (250×4.6 mm, 5 μm, Thermo-Fisher, MA, USA), under the detection conditions of 30°C, using a Shimadzu SPD-20A detector, the mobile phase is 20 mM ammonium acetate, containing 5% acetonitrile; flow rate: 1.0 mL / min; detection wavelength: 259 nm; column oven temperature: 30°C. Test content: After 45 minutes, the content of nicotinamide (NR), NAD derivatives and NR was measured; the test results are shown in Table 1:

[0126] Table 1

[0127] project NR / ppm NAMm / ppm NA / ppm Example 1 56291 160 359 Example 2 56302 158 355 Example 3 56289 159 356 Example 4 56295 161 360 Example 5 56298 158 358 Example 6 56305 159 357 Example 7 56296 160 356 Example 8 56293 161 359 Comparative Example 1 49255 253 468 Comparative Example 2 49248 248 437 Comparative Example 3 48972 241 444 Comparative Example 4 50041 268 487 Comparative Example 5 49358 251 467 Comparative Example 6 50074 230 418 Comparative Example 7 49781 246 467 Comparative Example 8 49479 261 454

[0128] As can be seen from Table 1, the yields of nicotinamide riboside prepared in Examples 1-8 are higher than those in Comparative Examples 1-8, and the by-products obtained are also lower than those in Comparative Examples 1-8. It can be seen that the process for producing nicotinamide riboside by high-density fermentation provided by the present invention has the advantages of high reaction yield and mild conditions.

[0129] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0130] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for producing nicotinamide riboside by high-density fermentation, characterized in that: The following steps are involved: Step 1, preparing culture medium and solution: preparing LB culture medium, fermentation medium, nicotinamide solution and glucose solution; Step 2: Prepare seed solution: Sterilize LB solid medium and spread it on a double-antibody plate for culture. Pick a single bacterium and streak it on a new double-antibody plate for further culture. Streak the strain and inoculate it on LB medium. Add Kan and Sm and continue to culture. OD 600 When the pH value is 1.5-2.5, the culture is terminated and the seed solution is obtained; Step 3, fermentation: inoculate the seed liquid into the fermentation tank, then add the fermentation medium, adjust the pH, maintain the temperature at 30-37°C, and keep the DO at 10-30%; after the DO rises and the glucose in the medium is consumed, add the glucose solution to maintain the glucose concentration in the fermentation medium <0.1g / L, and the OD 600 ≥15-20, add IPTG for induction, and after induction, add nicotinamide solution to maintain the nicotinamide content ≤0.5g / L to obtain fermentation liquid; Step 4, filtering the fermentation broth once and twice in sequence to obtain a filtrate; Step 5, adding the filtrate and water into a reaction kettle, the reaction temperature is 23-25°C, adding 3-cyanopyridine to react, and obtaining a hydrolyzate; Step 6: Ultrafiltration is performed on the hydrolyzate, followed by vacuum concentration, spray drying, and granulation to obtain nicotinamide riboside.

2. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: The LB medium in step 1 contains 5-6 g / L yeast extract powder, 8-10 g / L peptone, 10-11 g / L sodium chloride, and 15-18 g / L agar strips to prepare LB solid medium.

3. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: In step 1, the fermentation medium contains KH2PO4 6.67-6.70 g / L, citric acid 1.67-1.70 g / L, ammonium sulfate 1.50-1.55 g / L, glucose 10-20 g / L, MgSO4·7H2O 1.5-5 g / L, yeast powder 0.2-1 g / L, ferrous sulfate 0.05-0.1 g / L, manganese sulfate 0.05-0.1 g / L, CoCl2 0.001-0.002 g / L, VB1 0.01-0.02 g / L, VH 0.01-0.02 g / L, defoamer 0.01%, Kan 0.02 g / L, and ST 0.02 g / L.

4. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: The nicotinamide solution in step 1 is prepared by the following steps: Dissolve nicotinamide in pure water, place in a sterilizer, and sterilize at 121°C for 20 minutes; the dosage ratio of nicotinamide to pure water is 1g:10mL.

5. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: The glucose solution described in step 1 is prepared by the following steps: Dissolve glucose monohydrate in pure water, place in an autoclave, and sterilize at 115°C for 20 minutes; the dosage ratio of glucose monohydrate to pure water is 6g:10mL.

6. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: The final concentration of Kan and Sm in step 2 was 50 mg / L.

7. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: In step 3, the pH is adjusted to 6.7-7.2; the concentration of IPTG is 0.1-0.5 mM / L.

8. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: In step 4, the first filtration is performed by ultrafiltration, and the second filtration is performed by microfiltration; wherein the pore size of the ultrafiltration membrane is The pore size of the microfiltration membrane is 1-3 μm.

9. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: In step 5, the volume ratio of the filtrate to water is 1:(5-7); the volume ratio of the filtrate to 3-cyanopyridine is 1:

2.

10. The process for producing nicotinamide riboside by high-density fermentation according to claim 1, characterized in that: During the reduced pressure concentration, the temperature is 68-75°C, the pressure is 18-25KPa, and the time is 5-7h.