Method for improving shake-flask fermentation level of nicotinamide ribose

Abstract

The invention provides a method for improving the shake flask fermentation level of nicotinamide ribose, which comprises the following steps: S1, preparing a seed solution, namely coating a bacterial solution capable of producing nicotinamide riboside on an LB resistant plate of Kan and Sm, and culturing at 37 DEG C for 16 hours; picking a single colony, inoculating the single colony into an LB shake flask, adding Kan and Sm with the final concentration of 50mg / L, and culturing at 37 DEG C and 200rpm until OD600 is equal to 3-4; s2, fermentation culture of nicotinamide ribose: transferring the seed solution prepared in S1 into a fermentation culture medium according to the transferring amount of 4%, adding 50mg / L of Kan and Sm into the fermentation solution, and culturing at 37 DEG C and 220rpm for 1-4 hours; after the temperature is reduced to 30 DEG C, 0.1 g / L CoCl2 and 0.5 mM IPTG are added for induction, and 3.5 g / L nicotinamide is added 0-2 h after induction; kan and Sm are supplemented 9-15 hours after induction; nicotinamide ribose is collected to determine the culture time before induction and the substrate adding time after induction, so that the shake flask yield of the nicotinamide ribose is greatly increased. The corn steep liquor solution is properly added to provide growth factors and improve the glucose metabolism flux. According to the method, the problem that a large amount of plasmids are lost after induction is solved by additionally supplementing resistance, and the industrialization of nicotinamide ribose is accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of shake-flask fermentation, and specifically relates to a method for improving the shake-flask fermentation level of nicotinamide riboside. Background Art

[0002] Nicotinamide riboside (NR) is a type of vitamin B3 nutrient composed of nicotinamide and ribose. It naturally exists in yeast, bacteria, milk, and mammals and is a precursor and exogenous supplement of nicotinamide adenine dinucleotide (NAD+), an important coenzyme in oxidative phosphorylation and oxidative metabolism in the body. Supplementing nicotinamide riboside can increase the content of NAD+ and enhance the basic metabolic activities of cells, thereby significantly improving cell viability and various physiological functions of the human body.

[0003] There are mainly three categories of methods for producing nicotinamide riboside: chemical synthesis method, biocatalytic method, and microbial fermentation method. Currently, the chemical synthesis of nicotinamide riboside has advantages in terms of efficiency and cost. However, due to the toxic reagents and catalysts involved, this has greatly hindered its widespread application in the field of food safety. In contrast, as an environmentally friendly and sustainable production method, the microbial fermentation method cleverly utilizes the natural biosynthetic ability of microbial strains to produce nicotinamide riboside, providing a safer and greener option for the food industry. This method not only reduces the introduction of harmful substances but also demonstrates the great potential of biotechnology in modern food production. However, for the production of nicotinamide riboside by the microbial fermentation method reported currently, the fermentation unit is very low. For example, in the patent 201680078334.8, the yield is only about 0.1 - 100 mg / L; in the patent CN202210481044.5, the shake-flask yield is only 1.6 g / L, which limits its potential for large-scale commercial application.

[0004] The core objective of the present invention focuses on deeply optimizing the microbial fermentation conditions of nicotinamide riboside, aiming to significantly improve its production efficiency and yield, break through the bottleneck of the existing technology, and promote the development of nicotinamide riboside production towards a more efficient, more economical, and more environmentally friendly direction. Summary of the Invention

[0005] The core objective of the present invention focuses on deeply optimizing the microbial fermentation conditions of nicotinamide riboside, aiming to significantly improve its production efficiency and yield, break through the bottleneck of the existing technology, and promote the development of nicotinamide riboside production towards a more efficient, more economical, and more environmentally friendly direction. The present invention provides a method for improving the shake-flask fermentation level of nicotinamide riboside.

[0006] The specific implementation steps of this technical solution are as follows:

[0007] A method for improving the shake flask fermentation level of nicotinamide riboside, the method comprising the following steps:

[0008] S1. Preparation of seed liquid:

[0009] S1-1: Coating the bacterial liquid capable of producing nicotinamide riboside on an LB resistance plate containing streptomycin and kanamycin, and culturing at 37 °C for 16 h;

[0010] S1-2: Picking single colonies and inoculating them into an LB shake flask, adding streptomycin and kanamycin with a final concentration of 50 mg / L, and culturing at 37 °C

[0011] at 200 rpm until OD 600 = 3 - 4;

[0012] S2. Fermentation culture of nicotinamide riboside:

[0013] S2-1: Transferring the seed liquid prepared in S1 into the fermentation medium according to a transfer amount of 4%, adding streptomycin and kanamycin at 50 mg / L to the fermentation broth, and culturing at 37 °C and 220 rpm for 1 - 4 h;

[0014] S2-2: After reducing the temperature to 30 °C, adding 0.1 g / L CoCl2 and 0.5 mM IPTG for induction, and adding 3.5 g / L of nicotinamide 0 - 2 h after induction; adding streptomycin and kanamycin resistance 9 - 15 h after induction; collecting nicotinamide riboside.

[0015] Furthermore, the formula of the LB medium added in the LB resistance plate described in S1-1 and the LB shake flask described in S1-2 is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, and the addition amounts of streptomycin and kanamycin in the LB resistance plate described in S1-1 are both 50 mg / L respectively.

[0016] Furthermore, the formula of the fermentation medium described in S2-1 is: dipotassium hydrogen phosphate 15 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, corn steep liquor not less than 3 g / L, citric acid monohydrate 1 g / L, yeast extract 5 g / L, anhydrous glucose 200 g / L, magnesium sulfate heptahydrate 0.1 g / L.

[0017] Furthermore, in S2-1, culture at 37 °C and 220 rpm for 3 h.

[0018] Furthermore, add 3.5 g / L of nicotinamide 0.5 h after induction in S2-2.

[0019] Furthermore, add streptomycin and kanamycin resistance 9 hours after induction in S2-2.

[0020] Further, the added streptomycin and kanamycin resistance in S2-2 means that the added amounts of streptomycin and kanamycin in the system are 50 mg / L respectively.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. By determining the pre-induction culture time and the substrate addition time after induction, the shake flask yield of nicotinamide riboside is greatly increased.

[0023] 2. Appropriately adding corn steep liquor solution to provide growth factors and improve sugar metabolic flux.

[0024] 3. By additionally supplementing resistance, the present invention effectively solves the problems of plasmid stability decline and massive loss during the induction expression process, thereby significantly improving the final yield of the target product, nicotinamide riboside, and strongly promoting the industrialization process of nicotinamide riboside production. Accelerated the industrialization of nicotinamide riboside Description of the Drawings

[0025] Figure 1 Liquid chromatogram of nicotinamide riboside Detailed Embodiments

[0026] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0027] Step 1: Activate the strain and prepare the seed solution

[0028] S1-1: Take out the glycerol-preserved bacteria from the -80°C refrigerator (the recombinant strain NR011 obtained from the Chinese invention patent CN202210481044.5, a recombinant bacterium producing nicotinamide riboside), dilute it by 10 -4 times and coat it on an LB resistance (50 mg / L Kan and Sm) plate, and incubate it statically at 37°C for 16 h.

[0029] S1-2: Pick a single colony and inoculate it into an LB shake flask, add Kan and Sm with a final concentration of 50 mg / L, and culture it overnight at 37°C and 220 rpm for 13 h, and measure the OD600 to be approximately 3 - 4.

[0030] Step 2: Ferment and culture nicotinamide riboside

[0031] S2-1: Transfer the seed solution to 25 ml of fermentation medium according to a transfer amount of 4%, add 50 mg / L Kan and Sm to the fermentation broth, and then culture it at 37°C and 220 rpm for 1 - 4 h.

[0032] S2-2: Start induction after the temperature is reduced to 30°C. Add 0.1 g / L CoCl2 and 0.5 mM IPTG for induction, and add 3.5 g / L nicotinamide 0 - 2 h after induction. Supplement streptomycin and kanamycin resistance 9 - 15 h after induction so that the content of streptomycin and kanamycin in the system is 50 mg / L respectively; the fermentation cycle is 44 h.

[0033] The formula of the LB medium added to the LB resistance plate described in S1-1 and the LB shake flask described in S1-2 is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L. The addition amounts of streptomycin and kanamycin in the LB resistance plate described in S1-1 are 50 mg / L respectively.

[0034] The formula of the fermentation medium described in S2-1 is: dipotassium hydrogen phosphate 15 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, corn steep liquor 0 - 3 g / L, citric acid monohydrate 1 g / L, yeast extract 5 g / L, anhydrous glucose 200 g / L, magnesium sulfate heptahydrate 0.1 g / L.

[0035] The measurement and analysis methods involved in the examples:

[0036] 1. Calculation of conversion rate: Molar conversion rate (%) = (nicotinamide concentration ÷ 122) / (NR / 256) × 100%.

[0037] 2. Relative molecular mass of nicotinamide: 122; relative molecular mass of nicotinamide riboside: 256.

[0038] 3. Calculation of plasmid retention rate: Plasmid retention rate = number of colonies on the LB plate with kan and Sm resistance / number of colonies on the LB plate without antibiotic × 100%.

[0039] 4. Detection of the content of nicotinamide riboside

[0040] (1) Chromatographic conditions

[0041] Chromatographic column: Eclipse plus C18 4.6 * 250 mm 5um

[0042] Sample injection volume: 10 ul

[0043] Mobile phase: Phase A: 5 mmol (1 g / L) sodium heptanesulfonate, add 30 ul phosphoric acid, filter through membrane and ultrasonicate for later use

[0044] Phase B: pure methanol (HPLC grade), filter through membrane and ultrasonicate for later use.

[0045] Phase A: Phase B = 80:20

[0046] Detection wavelength: 259 nm

[0047] Flow rate: 0.8 ml / min

[0048] Column temperature: 30 °C

[0049] (2) Standard curve and sample treatment:

[0050] Standard curve: Weigh about 0.05 g of nicotinamide and nicotinamide riboside (NR) (accurate to 0.0001 g), dissolve it with ultrapure water, make up the volume, and shake well to obtain the stock solution.

[0051] Successively take six gradients of about 50 ppm, 100 ppm, 200 ppm, 300 ppm, 400 ppm, and 500 ppm of the stock solution, inject each gradient according to the chromatographic conditions, and make a standard curve.

[0052] Note: The preparation of the stock solution should be calculated according to the actual content of the substance. (For example, the weighed amount of NR is 0.05003 g, the effective content of NR is 95.026%, and the actual content should be 0.05003 * 0.95026 * 1000000 / 100 = 475.42 ppm).

[0053] Sample detection: Dilute the sample to the content value within the standard curve, inject it according to the chromatographic conditions, and obtain the content result = the content value displayed by the instrument * dilution factor.

[0054] Example 1: Optimization of the culture time before induction of the fermentation broth

[0055] Transfer the seed liquid to 25 ml of fermentation medium according to a transfer volume of 4%, add 50 mg / L Kan and Sm to the fermentation broth, and culture at 37 °C and 220 rpm for 1 h, 2 h, 3 h, and 4 h. After fermentation for 44 h, take samples for comparison and observe the results, as shown in Table 1:

[0056] Table 1 Results of shake flask fermentation

[0057]

[0058] As can be seen from Table 1, when the culture time before induction of the fermentation broth is 3 h, the yield of nicotinamide riboside is the highest, reaching 1.95 g / L. Therefore, in subsequent experiments, the culture time before induction will be controlled at 3 h.

[0059] Example 2: Optimization of the substrate addition time after induction of the fermentation broth

[0060] Transfer the seed liquid to 25 ml of fermentation medium according to a transfer volume of 4%, add 50 mg / L Kan and Sm to the fermentation broth, culture at 37 °C and 220 rpm for 3 h, then reduce the temperature to 30 °C and start induction, add 0.1 g / L CoCl2 and 0.5 mM IPTG, and add 3.5 g / L of nicotinamide at 0, 0.5 h, 1 h, and 2 h after induction. The fermentation results are shown in Table 2:

[0061] Table 2 Results of shake flask fermentation

[0062]

[0063] As can be seen from Table 2, after the fermentation broth was induced, the yield of the substrate nicotinamide added immediately was lower than that added 0.5 h after induction. As the addition time of nicotinamide was delayed, the OD value increased, but the OD value and the yield did not show a positive correlation. It is speculated that most of the glucose was utilized for cell growth rather than the production of NR.

[0064] Example 3: Additional supplementation of resistance

[0065] The seed liquid was transferred to 25 ml of fermentation medium at a transfer volume of 4%. After adding 50 mg / L Kan and Sm to the fermentation broth, it was cultured at 37 °C and 220 rpm for 3 h. After the temperature was lowered to 30 °C, induction was started, 0.1 g / L CoCl2 and 0.5 mM IPTG were added, and 3.5 g / L of nicotinamide was added 0.5 h after induction. Control group: no additional supplementation of resistance; Experimental group: additional supplementation of resistance once 9 h after induction. Starting from 5 h after induction, the plasmid retention rate was counted every two hours, and the statistical results are as follows:

[0066] Table 3 Plasmid retention rate of the control group

[0067]

[0068]

[0069] Table 4 Plasmid retention rate of the experimental group

[0070] Experimental group 5h 7h 9h 11h 13h 15h 17h 19h LB without antibiotics 878 998 704 640 685 959 1045 1183 <![CDATA[LB K + 、Sm + > 880 978 685 609 654 912 997 1126 Plasmid retention rate % 100.23 98.00 97.30 95.16 95.47 95.10 95.41 95.18

[0071] Table 5 Results of shake flask fermentation

[0072]

[0073] Supplementation of resistance 9 h after the fermentation broth was induced can improve plasmid stability, and the plasmid retention rate can still be maintained above 95% 19 h after induction. Without additional supplementation of resistance, plasmid loss is serious. And the shake flask results show that after adding resistance, the highest yield of nicotinamide ribose can reach 3.73 g / L, which is 34.66% higher than that of the control group.

[0074] Example 3: Optimization of the conditions for adding corn steep liquor solution

[0075] Inoculate the seed culture into 25 ml of fermentation medium at an inoculation volume of 4%. After adding 50 mg / L Kan and Sm to the fermentation broth, culture it at 37 °C and 220 rpm for 3 h. Then reduce the temperature to 30 °C and start induction. Add 0.1 g / L CoCl2, 0.5 mM IPTG, and add 3.5 g / L of nicotinamide 0.5 h after induction. Add the resistance once again 9 h after induction, and the fermentation cycle is 44 h.

[0076] The formula of the fermentation medium is as follows: dipotassium hydrogen phosphate 15 g / L, potassium dihydrogen phosphate 3 g / L, ammonium sulfate 5 g / L, corn steep liquor 0 - 3 g / L, citric acid monohydrate 1 g / L, yeast powder 5 g / L, anhydrous glucose 200 g / L, magnesium sulfate heptahydrate 0.1 g / L. The concentration gradient of corn steep liquor is 0 g / L, 1 g / L, 2 g / L, and 3 g / L. The fermentation results are shown in Table 6:

[0077] Table 6 Fermentation results in shake flasks

[0078]

[0079] As can be seen from Table 6, adding corn steep liquor solution to the fermentation medium is beneficial to the growth of bacteria, and can also supplement the growth factors required during the fermentation process, thereby increasing the yield of nicotinamide riboside. The highest amount of nicotinamide riboside detected by HPLC can reach 5.14 g / L after 44 h, and the results are as Figure 1 , with a molar conversion rate of 82%.

[0080] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the level of nicotinamide riboside shake flask fermentation, characterized in that: The method comprises the following steps: S1. Preparation of seed solution: S1-1: Spread the bacterial liquid capable of producing nicotinamide riboside on LB resistance plates containing streptomycin and kanamycin, and culture at 37°C for 16 h; S1-2: Pick a single colony and inoculate it into an LB shake flask, add streptomycin and kanamycin at a final concentration of 50 mg / L, and incubate at 37°C Cultivate at 200 rpm until OD 600 =3-4; S2. Fermentation and cultivation of nicotinamide riboside: S2-1: The seed solution prepared in S1 was transferred to the fermentation medium at a transfer rate of 4%, and 50 mg / L streptomycin and kanamycin were added to the fermentation broth, and cultured at 37°C and 220 rpm for 1-4 h; S2-2: After lowering the temperature to 30°C, add 0.1 g / L CoCl2 and 0.5 mM IPTG for induction, and add 3.5 g / L nicotinamide 0-2 h after induction; add streptomycin and kanamycin resistance 9-15 h after induction; collect nicotinamide riboside.

2. The method according to claim 1, characterized in that The LB culture medium formula added to the LB resistance plate described in S1-1 and the LB shake flask described in S1-2 is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, and the amount of streptomycin and kanamycin added to the LB resistance plate described in S1-1 is 50mg / L respectively.

3. The method according to claim 1, characterized in that The fermentation medium formula of S2-1 is: 15 g / L dipotassium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, not less than 3 g / L corn steep liquor, 1 g / L citric acid monohydrate, 5 g / L yeast powder, 200 g / L anhydrous glucose, and 0.1 g / L magnesium sulfate heptahydrate.

4. The method according to claim 1, characterized in that: The cells were cultured in S2-1 at 37°C and 220 rpm for 3 h.

5. The method according to claim 1, characterized in that S2-2 was added with nicotinamide (3.5 g / L) 0.5 h after induction.

6. The method according to claim 1, characterized in that Streptomycin and kanamycin resistance were added 9 hours after S2-2 induction.

7. The method according to claim 1, characterized in that The supplementation of streptomycin and kanamycin resistance in S2-2 is to make the amount of streptomycin and kanamycin added into the system be 50 mg / L respectively.

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