High efficiency process for isolation of the precursor of β-nicotinamide mononucleotide from fermentation broth + from fermentation broth

Abstract

The application discloses a high-efficiency method for separating and extracting a precursor beta-nicotinamide mononucleotide of NAD+ from a fermentation liquor. The method combines multi-stage membrane separation and boric acid affinity adsorption technology, and comprises the following steps: firstly, the fermentation liquor is sequentially pretreated through microfiltration, ultrafiltration and nanofiltration to effectively remove impurities such as bacteria, proteins and polysaccharides, and to preliminarily concentrate NMN; subsequently, the pH value of the NMN solution after membrane treatment is adjusted to 8.0-9.5, so that the NMN solution is suitable for specific combination with boric acid affinity resin; then, the NMN solution is adsorbed by using boric acid affinity resin (polystyrene-divinylbenzene or acrylate skeleton), and is eluted by using a specific eluent, and after post-treatment, the purified NMN product is obtained. The application effectively utilizes the synergistic effect of membrane separation and boric acid affinity adsorption, realizes high-efficiency and high-selectivity separation and purification of NMN, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] This application belongs to NAD + This invention relates to the field of precursor separation and purification technology, and in particular to a method for separating and extracting β-nicotinamide mononucleotide (NMN) from fermentation broth. Background Technology

[0002] Nicotinamide mononucleotide (NMN) is a naturally occurring bioactive nucleotide. NMN exists in two irregular forms, α and β. The β isomer is the active form of NMN, with a molecular weight of 334.221 g / mol. Its structural formula is as follows:

[0003]

[0004] NMN is mainly converted into NAD. + And thus, it plays a role. Among them, NAD... + Also known as coenzyme I, or nicotinamide adenine dinucleotide, it is widely distributed in all cells of the human body, participating in thousands of biocatalytic reactions, and is an essential coenzyme in the human body. NAD+ plays a crucial role in the aging process. + The decline in [certain substances] is considered a major cause of diseases and disabilities, such as hearing and vision loss, cognitive and motor dysfunction, immune deficiencies, arthritis caused by autoimmune inflammatory responses, metabolic disorders, and cardiovascular diseases.

[0005] Therefore, NMN supplementation increases NAD in the body. + The content of these substances can delay, improve, and prevent various aging-related phenotypes, or age-induced metabolic disorders and geriatric diseases.

[0006] Industrial preparation methods for NMN include chemical synthesis, hemienzymatic synthesis, holoenzymatic synthesis, and bio-fermentation. Among these, bio-fermentation has significant technological advantages in the industrial preparation of nicotinamide mononucleotide (NMN), mainly in the following aspects:

[0007] Green and environmentally friendly: Compared with chemical synthesis, bio-fermentation typically uses microorganisms as "cell factories" to carry out reactions under mild conditions. It does not require the use of large amounts of toxic and harmful chemical reagents, produces less waste, and is easy to handle, which meets the requirements of green industry and sustainable development.

[0008] Mild reaction conditions: Microbial fermentation generally takes place at room temperature, normal pressure, and near-neutral pH, which contrasts sharply with chemical synthesis methods that often require high temperature, high pressure, or extreme pH conditions. These milder reaction conditions reduce energy consumption, minimize equipment corrosion, and extend equipment lifespan.

[0009] High selectivity and high purity: Bioenzymes possess high specificity and selectivity. During microbial fermentation, enzymes can precisely catalyze specific reactions, reducing the formation of byproducts. This results in higher purity NMN, simplifies subsequent separation and purification steps, and reduces production costs.

[0010] Cost-effectiveness: Although the initial R&D investment in bio-fermentation may be high, once the strains and processes are optimized and mature, the raw material costs are relatively low, the production process has low energy consumption, and the purification costs are low, which is conducive to large-scale industrial production and reducing the price of the final product. Through genetic engineering and metabolic engineering, high-yield strains can be constructed to achieve efficient biosynthesis of NMN and can be continuously optimized.

[0011] Easy to scale up production: Fermentation tanks and other equipment are easy to scale up. Once the strain performance is stable and the fermentation process is mature, large-scale, continuous production can be achieved to meet the growing market demand for NMN.

[0012] Therefore, bio-fermentation has become a highly promising and competitive technology route in the industrial preparation of NMN due to its advantages such as being green and environmentally friendly, having mild reaction conditions, high selectivity, high purity, cost-effectiveness, and ease of large-scale production.

[0013] However, there are many types of impurities that may exist in the fermentation broth of NMN, which can be mainly divided into the following categories:

[0014] I. Residues, intermediates, and byproducts remaining during the production process: For example, nicotinamide riboside (NR). NMN and NR are structurally very similar, and NR is one of the precursors of NMN. If the enzyme conversion efficiency is low or side reactions occur during fermentation, NR may remain in the fermentation broth. Other examples include ATP (adenosine triphosphate), ADP (adenosine diphosphate), and AMP (adenosine monophosphate). These are important energy molecules and phosphate donors during fermentation and are also products of nucleotide metabolism. Other nucleotides and their derivatives: Complex nucleotide metabolic pathways exist in fermentation systems, which may produce various nucleotides with similar structures or properties to NMN, such as NAD+. + (Nicotinamide adenine dinucleotide), NADH, etc. NAD + It is a direct product of NMN. If NAD+ enzymes are present, NMN may be further converted into NAD. + Phosphates, such as sodium phosphate and potassium phosphate, are added to the fermentation broth as buffers or phosphorus sources, or are produced as metabolites. Sodium hexametaphosphate may also be added as an adjuvant or complexing agent in some processes.

[0015] II. Residual culture medium components: including but not limited to carbon sources, nitrogen sources (such as peptone, yeast extract, amino acids, etc., which may contain various small molecule peptides, amino acids, etc.), inorganic salts, etc.

[0016] III. Microbial Cells and Metabolic Products: Microbial Cells: After fermentation, the fermentation broth contains a large number of microbial cells (such as E. coli, yeast, and special engineered strains), which need to be removed by centrifugation, filtration, etc. Intracellular Substances: When cells lyse, large molecules such as proteins, nucleic acids (DNA, RNA), polysaccharides, and lipids are released into the fermentation broth. Extracellular Secretions: Metabolic products such as enzymes, pigments, organic acids, and extracellular polysaccharides secreted by microorganisms during their growth and metabolism are released into the culture medium. These substances may interfere with the purification of NMN and even affect its stability.

[0017] IV. Degradation Products: During fermentation or subsequent processing, NMN may be degraded due to factors such as pH, temperature, and enzyme activity, producing degradation products such as nicotinamide and ribose phosphate.

[0018] Therefore, there is an urgent need to develop a new process for efficiently separating NMN from NMN bio-fermentation broth. Summary of the Invention

[0019] The technical solution disclosed in this application aims to provide a new purification process that combines multi-stage membrane separation with boric acid affinity adsorption technology to achieve efficient and highly selective separation and purification of β-nicotinamide mononucleotide (NMN), which is suitable for large-scale industrial production.

[0020] To achieve the above objectives, this application adopts the following technical solution:

[0021] The first aspect of this application provides a method for separating and extracting NAD from fermentation broth. + A method for obtaining the precursor β-nicotinamide mononucleotide (NMN) includes the following steps:

[0022] S1: The fermentation broth containing NMN is microfiltered to obtain microfiltration permeate;

[0023] S2: The microfiltration permeate is subjected to ultrafiltration treatment to obtain ultrafiltration permeate;

[0024] S3: Perform nanofiltration treatment on the ultrafiltration permeate to obtain nanofiltration concentrate;

[0025] S4: Adjust the pH of the nanofiltration concentrate to 8.0 to 9.5;

[0026] S5: The nanofiltration concentrate after pH adjustment is adsorbed through boric acid affinity resin, so that NMN is adsorbed on the boric acid affinity resin;

[0027] S6: Elute the NMN adsorbed on the boric acid affinity resin with an eluent to obtain an NMN eluent.

[0028] It is worth noting that "fermentation broth containing NMN" refers to fermentation broth used to prepare NMN via bio-fermentation. This broth primarily contains: NMN product; substrates, intermediates, and byproducts remaining from the production process (e.g., NR, ATP, ADP, AMP); residual culture medium components (carbon source, nitrogen source, etc.); microbial cells and metabolites; and degradation products. In short, the composition of this fermentation broth is highly complex, thus requiring highly selective separation of NMN.

[0029] It is worth noting that, before step S1, in order to ensure the smooth progress of subsequent microfiltration operations, the fermentation broth containing NMN can undergo conventional pretreatment operations, including but not limited to: removing cells and large particulate impurities by centrifugation, preliminary filtration or flocculation sedimentation; adding anti-membrane clogging agents or performing pre-filtration, etc.

[0030] Preferably, in the above method, in step S1, the pore size of the microfiltration membrane is 0.1 μm to 0.45 μm; in step S2, the molecular weight cutoff (MWCO) of the ultrafiltration membrane is 1 kDa to 15 kDa; and in step S3, the molecular weight cutoff (MWCO) of the nanofiltration membrane is 100 Da to 700 Da. More preferably, the molecular weight cutoff (MWCO) of the nanofiltration membrane is 100-300 Da. The material of the microfiltration membrane can be polyvinylidene fluoride (PVDF), polyethersulfone (PES), ceramic membrane, etc.; considering the complexity of the fermentation broth, membrane materials resistant to fouling and acids / alkalis are more preferred. The material of the ultrafiltration membrane can be polyvinylidene fluoride (PVDF), polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), etc. The material of the nanofiltration membrane can be a polyamide composite membrane, etc.

[0031] Furthermore, in the process flow involved in the above method, step S1 aims to complete the microfiltration (MF) process, the purpose of which includes the preliminary removal of large particulate impurities such as bacterial cells, cell debris, and suspended particles from the fermentation broth, with NMN flowing out with the microfiltration permeate; step S2 aims to complete the ultrafiltration (UF) process, the purpose of which includes the further removal of large molecular weight proteins, polysaccharides, etc. from the microfiltration permeate, with NMN flowing out with the ultrafiltration permeate; step S3 aims to complete the nanofiltration (NF) process, the purpose of which includes the preliminary concentration and partial desalting of NMN in the ultrafiltration permeate, and the further removal of small molecular weight inorganic salts and some organic impurities, with NMN present in the nanofiltration concentrate.

[0032] Preferably, in the above method, the skeleton of the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) skeleton or an acrylate skeleton.

[0033] More preferably, in the above method, the skeleton of the boric acid affinity resin is polystyrene-divinylbenzene (St-DVB), and the degree of crosslinking of the boric acid affinity resin is 12% to 25% (e.g., 18% DVB).

[0034] It is worth explaining that, because the target product β-nicotinamide mononucleotide (NMN) contains a cis-vicinal diol structure, the boric acid (or its derivatives) in the boric acid affinity resin can form a reversible covalent ester bond with this structure under specific pH conditions, i.e., a borate ester. Furthermore, the polystyrene-divinylbenzene backbone possesses good mechanical strength and chemical stability, and is readily introduced with boric acid groups through functionalization. Moderately crosslinked St-DVB (e.g., 12% to 25% DVB) is preferred to provide sufficient porosity and mechanical strength while avoiding excessive hydrophobicity that could interfere with the affinity of boric acid. In addition, acrylate backbones (such as glycidyl methacrylate (GMA) and its copolymers) are generally more hydrophilic, which is beneficial for the activity of boric acid groups in the aqueous phase.

[0035] More preferably, in the above method, the boric acid groups grafted onto the backbone of the boric acid affinity resin are phenylboronic acid (PBA). Even more preferably, the amount of phenylboronic acid grafted onto the backbone of the boric acid affinity resin is 0.69-0.85 mmol / g per gram. Most preferably, the amount of phenylboronic acid grafted onto the backbone of the boric acid affinity resin is 0.79 mmol / g. The hydrophobicity of the benzene ring can synergistically interact with the hydrophilicity of boric acid (forming ester bonds), thereby contributing to the binding strength and pH responsiveness to the cis-ortho-diol structure. The inventors have experimentally discovered that a higher density of phenylboronic acid groups generally implies a higher adsorption capacity, but may lead to steric hindrance or affect elution efficiency to some extent. In view of this, the inventors unexpectedly discovered that the grafting amount of phenylboronic acid of 0.69 - 0.85 mmol / g can exhibit a high adsorption capacity and facilitate smooth elution, thus achieving both the goal of highly selective adsorption of NMN and the effect of efficient elution of the target product NMN.

[0036] Preferably, in the above method, after adjusting the pH value to 8.0 to 9.5 in step S4, the conductivity of the nanofiltration concentrate is less than 2.0 mS / cm.

[0037] To adjust the pH of the nanofiltration concentrate to the weakly alkaline environment required for boric acid affinity adsorption, step S4 was performed in the above method; in particular, controlling the conductivity to less than 2.0 mS / cm is very beneficial for subsequent boric acid affinity adsorption, because high ionic strength tends to weaken the binding of NMN to boric acid groups (which may affect the ionization equilibrium of boric acid or cause non-specific competition from other ions).

[0038] Preferably, in the above method, in step S5, the loading flow rate is 0.5 BV / h to 2 BV / h, typically performed at room temperature (e.g., 18-25°C). A more preferred loading flow rate is 0.9~1.6 BV / h. This suitable loading flow rate facilitates sufficient contact between the cis-vicinal diol structure of the NMN ribose moiety and the borate groups on the borate affinity resin, forming a reversible covalent bond, thereby specifically adsorbing NMN. After loading, NMN is adsorbed onto the borate affinity resin, and the waste liquid (a solution without NMN) is discharged.

[0039] After sample loading and before step S6, an additional washing step is required. This washing step aims to remove non-specific adsorbed impurities. The washing buffer used is a buffer solution with the same pH as the adsorbent (pH 8.0-9.5), the washing volume is 3-5 column volumes (BV), the washing flow rate is 1-3 BV / h, and it is usually performed at room temperature (e.g., 18-25°C). Preferably, the buffer solution that can be used as the washing buffer is selected from any of the following: borate buffer, HEPES buffer, CHES buffer, and CAPSO buffer.

[0040] Preferably, in the above method, in step S6, the eluent is a solution containing a competing ligand.

[0041] More preferably, in step S6, the competitive ligand is selected from one or more of the following: glycerol, erythritol, xylitol, ribose, arabinose, trehalose, and maltitol.

[0042] Preferably, in the above method, in step S6, the elution temperature is 18~25°C.

[0043] More preferably, in step S6, the concentration of the competing ligand is from 0.09 M to 0.4 M.

[0044] Regarding step S6, the following points should be noted:

[0045] Step S6 aims to dissociate NMN from the resin, thereby eluting NMN to obtain crude NMN. The competitive ligand is an added compound containing a cis-vicinal diol structure, which competitively binds to the borate group of NMN, thus promoting NMN elution.

[0046] It is worth noting that the inventors unexpectedly discovered that erythritol exhibits the strongest specific elution effect due to its specific structure, especially the two pairs of vicinal diols, with an elution rate >90%. Further experiments confirmed that the suitable concentration of erythritol is 0.1–0.2M, the elution volume is 3–5 BV, and the elution temperature is 20–25℃.

[0047] In addition, after step S6, there are conventional post-processing steps for refining the NMN product to a high purity. For example, these post-processing steps may include operations such as concentration, recrystallization, and spray drying of the NMN eluent, which will not be described in detail herein.

[0048] Furthermore, it is worth mentioning that in the above method, the eluted resin undergoes regeneration / activation treatment for reuse in the next batch of adsorption, thus achieving recycling. For example, it can be washed with borate buffer solution or dilute alkaline solution (such as dilute NaOH) with a pH of 8.0-9.5, followed by washing with pure water until equilibrium is reached. Similarly, since the above regeneration / activation treatment is a routine choice made by researchers according to actual production needs, it will not be elaborated upon in this article.

[0049] In summary, this application provides a method for separating and extracting NMN from fermentation broth. This method achieves efficient and highly selective separation and purification of NMN by combining multi-stage membrane separation technology (microfiltration, ultrafiltration, nanofiltration) with boric acid affinity adsorption technology.

[0050] In summary, compared with the prior art, the technical solution provided in this application has at least the following beneficial effects:

[0051] (1) Significantly improved NMN separation and purification efficiency and selectivity: This application utilizes a three-stage membrane separation combination of "microfiltration-ultrafiltration-nanofiltration" to precisely remove impurities of different molecular weights (such as bacterial cells, cell debris, large molecular proteins, polysaccharides, and some small molecular inorganic salts and organic impurities) step by step during the fermentation broth pretreatment stage, providing a purer feed solution for subsequent affinity adsorption. This not only reduces the burden on boric acid affinity resin but also minimizes the interference of impurities on NMN adsorption. The subsequent boric acid affinity adsorption technology utilizes the unique cis-vicinal diol structure in the NMN molecule and its specific reversible covalent bond with the boric acid group to achieve highly selective adsorption of NMN in complex systems, effectively distinguishing NMN from structurally similar impurities (such as NR, ATP, ADP, etc.). This multi-stage membrane separation technology combined with boric acid affinity adsorption technology significantly improves the total yield of NMN and the purity of the final product.

[0052] (2) The boric acid affinity adsorption process conditions were optimized to ensure efficient adsorption and elution:

[0053] ① Before affinity adsorption, the pH of the nanofiltration concentrate was precisely adjusted to 8.0-9.5 and the conductivity was controlled to be less than 2.0 mS / cm, creating a weakly alkaline environment most conducive to the binding of NMN and boric acid groups, while avoiding the negative impact of high ionic strength on binding, thereby improving the adsorption capacity and binding strength of NMN.

[0054] ② The skeleton, degree of crosslinking, and amount of phenylboronic acid grafting of the boric acid affinity resin were optimized (e.g., polystyrene-divinylbenzene skeleton, 12%-25% DVB crosslinking degree, and 0.69-0.85 mmol / g phenylboronic acid grafting amount). This ensured that the resin had good mechanical strength, chemical stability, and optimal adsorption performance. In particular, the optimized amount of phenylboronic acid grafting (most preferably 0.79 mmol / g) not only ensured high adsorption capacity but also benefited subsequent elution efficiency.

[0055] ③ By introducing competitive ligands (such as erythritol) for elution, and utilizing the principle that erythritol competes with NMN to bind to the borate group, efficient and gentle elution of NMN is achieved. Among them, the inventors found that the optimal erythritol has excellent elution effect (elution rate >90%), and by controlling its concentration and elution temperature, the elution efficiency was further optimized and the consumption of eluent was reduced.

[0056] (3) Strong process integration and promising prospects for industrial application: This application organically combines mature membrane separation technology with affinity adsorption technology to form a continuous, efficient and easily scaled-up production process. Membrane separation operation is easy to automate and continuously produce, while affinity adsorption column chromatography also has a good scale-up effect. The entire process is mild and energy consumption is low, reducing equipment wear under high temperature, high pressure or extreme pH conditions. In addition, boric acid affinity resin can be regenerated and recycled, which further reduces production costs and conforms to the concept of green and environmentally friendly industrial production. This provides a highly competitive solution for the large-scale industrial preparation of NMN and is therefore suitable for large-scale industrial production. Attached Figure Description

[0057] Figure 1 The flowchart is a process for separating and extracting NMN from fermentation broth according to this application;

[0058] Figure 2 The 1H NMR spectrum of the purified NMN product. Detailed Implementation

[0059] The present application will now be described in detail with reference to the embodiments. Obviously, the described embodiments are merely some, and not all, of the embodiments of the present application. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative effort are within the scope of protection of the present application.

[0060] According to the technical solution provided in the first aspect of this application, a method for separating and extracting β-nicotinamide mononucleotide (NMN) from fermentation broth specifically includes the following steps:

[0061] S1: The fermentation broth containing NMN is microfiltered to obtain microfiltration permeate;

[0062] S2: The microfiltration permeate is subjected to ultrafiltration treatment to obtain ultrafiltration permeate;

[0063] S3: Perform nanofiltration treatment on the ultrafiltration permeate to obtain nanofiltration concentrate;

[0064] S4: Adjust the pH of the nanofiltration concentrate to 8.0 to 9.5;

[0065] S5: The nanofiltration concentrate after pH adjustment is adsorbed through boric acid affinity resin, so that NMN is adsorbed on the boric acid affinity resin;

[0066] S6: Elute the NMN adsorbed on the boric acid affinity resin with an eluent to obtain an NMN eluent.

[0067] In a preferred embodiment, in step S1, the pore size of the microfiltration membrane is 0.1 μm to 0.45 μm; in step S2, the molecular weight cutoff (MWCO) of the ultrafiltration membrane is 1 kDa to 15 kDa; and in step S3, the molecular weight cutoff (MWCO) of the nanofiltration membrane is 100 Da to 700 Da.

[0068] In a further preferred embodiment, the nanofiltration membrane has a molecular weight cutoff (MWCO) of 100-300 Da.

[0069] In a preferred embodiment, the boric acid affinity resin has a polystyrene-divinylbenzene (St-DVB) backbone or an acrylate backbone.

[0070] In a further preferred embodiment, the borate affinity resin has a backbone of polystyrene-divinylbenzene (St-DVB) and the degree of crosslinking of the borate affinity resin is 12% to 25%.

[0071] In a further preferred embodiment, the borate affinity resin has a backbone of polystyrene-divinylbenzene (St-DVB) and a crosslinking degree of 18%.

[0072] In some preferred embodiments, the boric acid groups grafted onto the backbone of the boric acid affinity resin are phenylboronic acid.

[0073] In a further preferred embodiment, the grafting amount of phenylboronic acid is 0.69 - 0.85 mmol / g.

[0074] In a most preferred embodiment, the grafting amount of phenylboronic acid is 0.79 mmol / g.

[0075] In a preferred embodiment, after adjusting the pH value to 8.0 to 9.5 in step S4, the conductivity of the nanofiltration concentrate is less than 2.0 mS / cm.

[0076] In some preferred embodiments, in step S5, the sample loading rate is 0.5 BV / h to 2 BV / h.

[0077] In some further preferred embodiments, the loading flow rate is 0.9~1.6 BV / h.

[0078] In a preferred embodiment, in step S6, the eluent is a solution containing a competing ligand.

[0079] In some further preferred embodiments, in step S6, the competing ligand is selected from one or more of the following: glycerol, erythritol, xylitol, ribose, arabinose, trehalose, and maltitol.

[0080] In a preferred embodiment, the competing ligand is erythritol.

[0081] In a preferred embodiment, in step S6, the elution temperature is 18~25°C.

[0082] In a further preferred embodiment, in step S6, the concentration of the competing ligand is from 0.09 M to 0.4 M.

[0083] The present application will be described in detail below through specific embodiments to enable a better understanding of the present application, but the following embodiments do not limit the scope of the present application.

[0084] Unless otherwise specified, the steps in the following examples are standard operations, and the reaction raw materials and reagents used are all available from publicly available commercial sources.

[0085] Example 1

[0086] Main raw materials and equipment

[0087] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0088] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0089] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0090] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0091] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0092] Main operating steps

[0093] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0094] S1 Microfiltration

[0095] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0096] S2 Ultrafiltration

[0097] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0098] S3 Nanofiltration

[0099] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0100] S4 pH and Conductivity Adjustment

[0101] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 8.0; and test the conductivity to be ≤1.8 mS / cm (meeting the requirement of <2.0 mS / cm).

[0102] S5 Boric acid affinity adsorption

[0103] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0104] After adsorption, wash with borate buffer at pH 8.0 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0105] S6 Competitive Elution

[0106] Elute with 0.16M maltitol solution (pH 8.0) at a flow rate of 1.5 BV / h, and collect the eluent (3 BV).

[0107] Elution temperature: 25℃, the measured NMN elution rate was 87%.

[0108] Post-processing and test results

[0109] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0110] Product Analysis:

[0111] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 71% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0112] Resin Regeneration

[0113] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 8.0 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0114] Example 2

[0115] Main raw materials and equipment

[0116] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0117] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0118] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0119] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0120] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0121] Main operating steps

[0122] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0123] S1 Microfiltration

[0124] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0125] S2 Ultrafiltration

[0126] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0127] S3 Nanofiltration

[0128] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0129] S4 pH and Conductivity Adjustment

[0130] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 8.3; and test the conductivity ≤ 1.8 mS / cm (meeting the requirement of < 2.0 mS / cm).

[0131] S5 Boric acid affinity adsorption

[0132] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0133] After adsorption, wash with borate buffer at pH 8.3 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0134] S6 Competitive Elution

[0135] Elute with 0.16M maltitol solution (pH 8.3) at a flow rate of 1.5 BV / h, and collect the eluent (3 BV).

[0136] Elution temperature: 25℃, the measured NMN elution rate was 85%.

[0137] Post-processing and test results

[0138] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0139] Product Analysis:

[0140] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 74% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0141] Resin Regeneration

[0142] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 8.3 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0143] Example 3

[0144] Main raw materials and equipment

[0145] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0146] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0147] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0148] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0149] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0150] Main operating steps

[0151] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0152] S1 Microfiltration

[0153] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0154] S2 Ultrafiltration

[0155] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0156] S3 Nanofiltration

[0157] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0158] S4 pH and Conductivity Adjustment

[0159] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 8.6; and test the conductivity ≤ 1.8 mS / cm (meeting the requirement of < 2.0 mS / cm).

[0160] S5 Boric acid affinity adsorption

[0161] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0162] After adsorption, wash with borate buffer at pH 8.6 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0163] S6 Competitive Elution

[0164] Elute with 0.16M maltitol solution (pH 8.6) at a flow rate of 1.5 BV / h, and collect the eluent (3 BV).

[0165] Elution temperature: 25℃, the measured NMN elution rate was 88%.

[0166] Post-processing and test results

[0167] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0168] Product Analysis:

[0169] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 75% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0170] Resin Regeneration

[0171] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 8.6 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0172] Example 4

[0173] Main raw materials and equipment

[0174] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0175] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0176] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0177] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0178] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0179] Main operating steps

[0180] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0181] S1 Microfiltration

[0182] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0183] S2 Ultrafiltration

[0184] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0185] S3 Nanofiltration

[0186] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0187] S4 pH and Conductivity Adjustment

[0188] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 9.0; and test the conductivity to be ≤1.8 mS / cm (meeting the requirement of <2.0 mS / cm).

[0189] S5 Boric acid affinity adsorption

[0190] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0191] After adsorption, wash with borate buffer at pH 9.0 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0192] S6 Competitive Elution

[0193] Elute with 0.16M maltitol solution (pH 9.0) at a flow rate of 1.5 BV / h, and collect the eluent (3 BV).

[0194] Elution temperature: 25℃, the measured NMN elution rate was 87%.

[0195] Post-processing and test results

[0196] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0197] Product Analysis:

[0198] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 78% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0199] Resin Regeneration

[0200] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 9.0 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0201] Example 5

[0202] Main raw materials and equipment

[0203] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0204] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0205] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0206] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0207] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0208] Main operating steps

[0209] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0210] S1 Microfiltration

[0211] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0212] S2 Ultrafiltration

[0213] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0214] S3 Nanofiltration

[0215] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0216] S4 pH and Conductivity Adjustment

[0217] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 9.1; and test the conductivity to be ≤1.8 mS / cm (meeting the requirement of <2.0 mS / cm).

[0218] S5 Boric acid affinity adsorption

[0219] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0220] After adsorption, wash with borate buffer at pH 9.1 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0221] S6 Competitive Elution

[0222] Elute with 0.16M maltitol solution (pH 9.1) at a flow rate of 1.5 BV / h, and collect the eluent (3 BV).

[0223] Elution temperature: 25℃, the measured NMN elution rate was 86%.

[0224] Post-processing and test results

[0225] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0226] Product Analysis:

[0227] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 73% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0228] Resin Regeneration

[0229] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 9.1 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0230] Example 6

[0231] Main raw materials and equipment

[0232] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0233] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0234] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0235] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0236] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.70 mmol / g.

[0237] Main operating steps

[0238] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0239] S1 Microfiltration

[0240] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0241] S2 Ultrafiltration

[0242] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0243] S3 Nanofiltration

[0244] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0245] S4 pH and Conductivity Adjustment

[0246] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 9.5; and test the conductivity ≤ 1.8 mS / cm (meeting the requirement of < 2.0 mS / cm).

[0247] S5 Boric acid affinity adsorption

[0248] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0249] After adsorption, wash with borate buffer at pH 9.5 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0250] S6 Competitive Elution

[0251] Elute with 0.16M maltitol solution (pH 9.5) at a flow rate of 1.5 BV / h, and collect the eluent (volume 3 BV).

[0252] Elution temperature: 25℃, the measured NMN elution rate was 85%.

[0253] Post-processing and test results

[0254] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0255] Product Analysis:

[0256] HPLC analysis showed the NMN product purity to be 95.6%; the overall yield was 70% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0257] Resin Regeneration

[0258] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 9.5 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0259] Based on the experimental data from Examples 1-6, the effect of the pH adjustment value in step S4 on the total yield of NMN is shown in Table 1 below:

[0260] Table 1

[0261]

[0262] Based on Examples 1-6 above, it can be seen that the pH adjustment value involved in step S4 and the related pH value in subsequent steps have a certain impact on the total yield of NMN. Specifically, the total yield of Examples 1-6 first increases and then decreases, with a peak total yield of 78%. At this time, the corresponding optimal pH value is 9.0 (Example 4).

[0263] Example 7

[0264] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.72 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating steps in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0265] Post-processing and test results

[0266] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0267] Product Analysis:

[0268] HPLC analysis showed the NMN product purity to be 96.0%; the overall yield was 79.5% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0269] Example 8

[0270] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.75 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating steps in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0271] Post-processing and test results

[0272] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0273] Product Analysis:

[0274] HPLC analysis showed the NMN product purity to be 96.7%; the overall yield was 80.1% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0275] Example 9

[0276] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.78 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating step in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0277] Post-processing and test results

[0278] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0279] Product Analysis:

[0280] HPLC analysis showed the NMN product purity to be 97.2%; the overall yield was 80.4% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0281] Example 10

[0282] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.79 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating steps in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0283] Post-processing and test results

[0284] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0285] Product Analysis:

[0286] HPLC analysis showed the NMN product purity to be 98.5%; the overall yield was 81.3% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0287] Example 11

[0288] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.82 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating steps in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0289] Post-processing and test results

[0290] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0291] Product Analysis:

[0292] HPLC analysis showed the NMN product purity to be 97.1%; the overall yield was 80.0% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0293] Example 12

[0294] In this embodiment, the boric acid affinity resin is a polystyrene-divinylbenzene (St-DVB) backbone with a crosslinking degree of 18% DVB and a phenylboronic acid grafting amount of 0.85 mmol / g. Apart from this, the process parameters of each raw material, equipment, and main operating steps in the process of separating and extracting NMN from the fermentation broth are the same as those in Example 4.

[0295] Post-processing and test results

[0296] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0297] Product Analysis:

[0298] HPLC analysis showed the NMN product purity to be 96.9%; the overall yield was 79.8% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0299] Table 2 below compares the key process parameters and test results of Examples 7-12, focusing on the impact of phenylboronic acid grafting amount on NMN purity and total yield:

[0300] Table 2

[0301]

[0302] Examples 7-12 systematically investigated the effect of the amount of phenylboronic acid grafted onto the boric acid affinity resin on the NMN separation and extraction efficiency while keeping the pH value at 9.0 (based on the best results from Examples 1-6) and other process parameters constant.

[0303] As shown in Table 2 above, the total yield and product purity of NMN first increased and then decreased with the increase of phenylboronic acid grafting amount. Specifically, when the phenylboronic acid grafting amount was 0.79 mmol / g (Example 10), the total yield and product purity of NMN reached the highest levels, at 81.3% and 98.5%, respectively. This indicates that at this grafting amount, the adsorption capacity and selectivity of the boric acid affinity resin for NMN reached the optimal balance. Lower grafting amounts (such as 0.72 mmol / g) may lead to insufficient adsorption sites, affecting the yield; while excessively high grafting amounts (such as 0.82 mmol / g and 0.85 mmol / g) may lead to enhanced steric hindrance effect, or affect the effective binding of NMN and subsequent elution efficiency, thereby slightly reducing the total yield and purity.

[0304] Example 13

[0305] Main raw materials and equipment

[0306] Fermentation broth: Escherichia coli engineered bacteria fermentation broth (NMN concentration 0.8 g / L, containing bacterial cells, protein, NR, inorganic salts and other impurities)

[0307] Microfiltration membrane: PES membrane, average pore size 0.25 μm

[0308] Ultrafiltration membrane: PVDF membrane, molecular weight cutoff 6~10 kDa

[0309] Nanofiltration membrane: Polyamide composite membrane, molecular weight cutoff 100-300 Da

[0310] Boric acid affinity resin: polystyrene-divinylbenzene (St-DVB) backbone, crosslinking degree 18% DVB, phenylboronic acid grafting amount 0.79 mmol / g.

[0311] Main operating steps

[0312] according to Figure 1 The flowchart shown illustrates the steps for separating and extracting NMN from the fermentation broth:

[0313] S1 Microfiltration

[0314] The fermentation broth was centrifuged at 8,000 rpm for 30 min at room temperature, and the supernatant was collected. The supernatant was then filtered through a PES microfiltration membrane at an operating pressure of approximately 0.15 MPa to obtain the microfiltration permeate.

[0315] S2 Ultrafiltration

[0316] The microfiltration permeate is passed through a PVDF ultrafiltration membrane at an operating pressure of approximately 0.4 MPa, and the ultrafiltration permeate is collected.

[0317] S3 Nanofiltration

[0318] The ultrafiltration permeate was passed through a polyamide nanofiltration membrane at an operating pressure of approximately 1.0 MPa, and the nanofiltration concentrate was collected (NMN concentration increased to 6.2 g / L).

[0319] S4 pH and Conductivity Adjustment

[0320] Add 1 M NaOH solution to the nanofiltration concentrate to make the pH 9.0; and test the conductivity to be ≤1.8 mS / cm (meeting the requirement of <2.0 mS / cm).

[0321] S5 Boric acid affinity adsorption

[0322] The adjusted nanofiltration concentrate was passed through a boric acid affinity resin column (2500 mL bed volume) at a flow rate of 1.2 BV / h and adsorbed at room temperature (25 °C).

[0323] After adsorption, wash with borate buffer at pH 9.0 for 4 BV (flow rate 2 BV / h) to remove non-specific impurities.

[0324] S6 Competitive Elution

[0325] Elute with 0.16M xylitol solution (pH 9.0) at a flow rate of 1.5 BV / h, and collect the eluent (volume 3 BV).

[0326] Elution temperature: 25℃, the measured NMN elution rate was 88%.

[0327] Post-processing and test results

[0328] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0329] Product Analysis:

[0330] HPLC analysis showed the NMN product purity to be 98.0%; the overall yield was 82.8% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0331] Resin Regeneration

[0332] The resin column was rinsed with 0.1 M NaOH for 2 BV, and then equilibrated with pH 9.0 borate buffer for 3 BV. It can be reused 10 times and the adsorption capacity remains >90%.

[0333] Example 14

[0334] In this embodiment, an arabinose solution was used instead of the xylitol solution in Example 13; the elution temperature was 25°C, and the NMN elution rate was measured to be 87%.

[0335] Apart from this, the process parameters for each raw material, equipment, and main operating step in the process of separating and extracting NMN from the fermentation broth are the same as in Example 13.

[0336] Post-processing and test results

[0337] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0338] Product Analysis:

[0339] HPLC analysis showed the NMN product purity to be 98.1%; the overall yield was 83.9% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0340] Example 15

[0341] In this embodiment, trehalose solution was used instead of xylitol solution in Example 13; elution temperature: 25°C, and the NMN elution rate was measured to be 88%.

[0342] Apart from this, the process parameters for each raw material, equipment, and main operating step in the process of separating and extracting NMN from the fermentation broth are the same as in Example 13.

[0343] Post-processing and test results

[0344] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0345] Product Analysis:

[0346] HPLC analysis showed the NMN product purity to be 98.0%; the overall yield was 84.7% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0347] Example 16

[0348] In this embodiment, erythritol solution was used instead of xylitol solution in Example 13; elution temperature: 25°C, and the NMN elution rate was measured to be 91%.

[0349] Apart from this, the process parameters for each raw material, equipment, and main operating step in the process of separating and extracting NMN from the fermentation broth are the same as in Example 13.

[0350] Post-processing and test results

[0351] The NMN eluent was concentrated to 1 / 10 of its original volume under reduced pressure at 40°C. Then, 4 times the volume of anhydrous ethanol was added, and the mixture was crystallized at about 5°C for 12 hours. The crystals were then filtered to obtain white crystals.

[0352] Product Analysis:

[0353] HPLC analysis showed the NMN product purity to be 98.6%; the overall yield was 85.0% (calculated from the fermentation broth); the NMN product's 1H NMR spectrum is as follows. Figure 2 As shown.

[0354] Table 3 below summarizes the elution solutions and corresponding test results for Examples 13-16 (all other process parameters are the same):

[0355] Table 3

[0356]

[0357] Therefore, based on the test results of Examples 13-16, it can be seen that erythritol solution successfully improved the total yield by increasing elution efficiency and reducing impurity residue, and is considered the most preferred competitive ligand.

[0358] In summary, this application provides a novel method for the efficient separation and extraction of NMN from fermentation broth. By combining multi-stage membrane separation and boric acid affinity adsorption technology, highly efficient and selective purification of NMN is achieved. This method not only significantly improves the yield and purity of NMN by optimizing pH and key resin parameters (backbone structure, degree of crosslinking, and amount of phenylboronic acid grafting), but also introduces specific competitive ligands to achieve efficient elution. This integrated process offers advantages such as being mild, environmentally friendly, and easily scalable, providing an innovative and competitive solution for the industrial preparation of NMN.

[0359] The specific embodiments of this application have been described in detail above, but they are merely examples, and this application is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this application are also within the scope of this application. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of this application should be covered within the scope of this application.

Claims

1. Separation and extraction of NAD from fermentation broth + An efficient method for the precursor β-nicotinamide mononucleotide, characterized in that, Includes the following steps: S1: The fermentation broth containing NMN is microfiltered to obtain microfiltration permeate; S2: The microfiltration permeate is subjected to ultrafiltration treatment to obtain ultrafiltration permeate; S3: Perform nanofiltration treatment on the ultrafiltration permeate to obtain nanofiltration concentrate; S4: Adjust the pH of the nanofiltration concentrate to 8.0 to 9.5; S5: The nanofiltration concentrate after pH adjustment is adsorbed through boric acid affinity resin, so that NMN is adsorbed on the boric acid affinity resin; S6: Elute the NMN adsorbed on the boric acid affinity resin with an eluent to obtain an NMN eluent; The eluent is a solution containing a competitive ligand; the competitive ligand is selected from one or more of the following: erythritol, xylitol, arabinose, and trehalose.

2. The method according to claim 1, characterized in that, In step S1, the pore size of the microfiltration membrane is 0.1 μm to 0.45 μm; in step S2, the molecular weight cutoff (MWCO) of the ultrafiltration membrane is 1 kDa to 15 kDa; and in step S3, the molecular weight cutoff (MWCO) of the nanofiltration membrane is 100 Da to 700 Da.

3. The method according to claim 1, characterized in that, The boric acid affinity resin has a polystyrene-divinylbenzene (St-DVB) backbone or an acrylate backbone.

4. The method according to claim 3, characterized in that, The boric acid affinity resin has a skeleton of polystyrene-divinylbenzene (St-DVB) and a crosslinking degree of 12% to 25%.

5. The method according to claim 1, characterized in that, In step S4, after adjusting the pH value to 8.0 to 9.5, the conductivity of the nanofiltration concentrate is less than 2.0 mS / cm.

6. The method according to claim 1, characterized in that, In step S5, the sample loading rate is 0.5 BV / h to 2 BV / h.

7. The method according to claim 1, characterized in that, In step S6, the elution temperature is 18~25℃.

8. The method according to claim 1, characterized in that, In step S6, the concentration of the competing ligand is from 0.09 M to 0.4 M.

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