A microbial fermentation nutrient solution rich in beta-nicotinamide mononucleotide and preparation and application thereof

Abstract

The present application relates to the field of agricultural planting technology, and provides a kind of microorganism fermentation nutrient solution rich in beta-nicotinamide mononucleotide and its preparation and application, including microorganism fermentation nutrient solution, microorganism fermentation nutrient solution is obtained by filtering the upper clear liquid after the fermentation substrate is subjected to segmented fermentation by composite microbial inoculant;The composite microbial inoculant is composed of the following weight parts of strains: bacillus subtilis 20-30 parts, lactobacillus plantarum 25-35 parts, yeast 15-25 parts, bacillus megaterium 10-20 parts;The fermentation substrate is composed of the following components in mass percent: glucose 1.5%-3%, soybean meal hydrolysate 2%-4%, nicotinamide 0.08%-0.15%, magnesium sulfate 0.02%, potassium dihydrogen phosphate 0.03%, and the balance is water.The present application solves the problem that the prior art cannot produce high-content NMN functional nutrient solution at low cost through biological fermentation.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, and in particular to a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide and its preparation and application. Background Technology

[0002] Oxidized nicotinamide adenine dinucleotide (NAD) + NAD+ is a core coenzyme in plant and animal cells, widely involved in key physiological processes such as energy metabolism, antioxidant defense, stress response, and regulation of cellular senescence. During the plant growth cycle, abiotic stresses such as high temperature, drought, soil compaction, and continuous cropping obstacles continuously deplete endogenous NAD+ within cells. + Excessive NAD content leads to premature aging of plants, reduced fruit set rate, and decreased fruit quality. Therefore, increasing endogenous NAD in plant cells is crucial. + It is an important strategy to enhance crop resistance to stress, delay senescence, and improve yield and quality.

[0003] Currently, direct exogenous spraying of NAD + There is a significant defect: NAD + Due to their large molecular weight, NAD+ has difficulty penetrating plant epidermal cell membranes, resulting in extremely low absorption efficiency; furthermore, NAD+... + It is readily hydrolyzed by phosphatases in liquid environments, decomposes rapidly upon exposure to light and heat, and exhibits poor stability when stored for field application; furthermore, industrially purified NAD... + The cost of raw materials is extremely high.

[0004] β-Nicotinamide mononucleotide (NMN) is a precursor to NAD+. + It is a direct precursor of NAD+, possessing advantages such as small molecular weight, good water solubility, and strong stability. It can be directly absorbed by plant roots and leaves, and after entering cells, it is rapidly converted into endogenous NAD+. + Recent studies have shown that exogenous NMN treatment can effectively enhance plant disease resistance, stress resistance, and delay aging. However, currently commercially available NMN products are mainly produced using chemical synthesis or enzymatic catalysis, which are costly and leave chemical residues, making them unsuitable for organic farming.

[0005] Microbial fermentation for NMN production is a relatively new and emerging field. Current research often employs genetically engineered strains (such as recombinant E. coli and recombinant yeast) to increase NMN yield. However, the application of these genetically engineered strains in agriculture faces challenges related to biosafety compliance and large-scale production limitations. Furthermore, existing agricultural microbial fermentation nutrient solutions primarily enrich conventional metabolites such as amino acids, organic acids, and polysaccharides. There is a lack of complex strain formulations and fermentation processes capable of selectively enriching high levels of NMN, making it impossible to produce high-NMN functional nutrient solutions at low cost through biofermentation and effectively enhance endogenous NAD in plants. + level. Summary of the Invention

[0006] Therefore, to address the aforementioned problems, this invention proposes a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide, its preparation, and its application. Using safe, non-genetically modified strains, and through optimized composite strains and segmented fermentation processes, a low-cost microbial fermentation nutrient solution rich in high levels of NMN is prepared. Furthermore, the application of this microbial fermentation nutrient solution significantly increases endogenous NAD in plant cells. + It enhances resistance to adverse conditions, delays premature aging, improves soil quality, and enhances the quality of fruits and vegetables.

[0007] To solve this technical problem, the present invention adopts the following solution: a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide, comprising a microbial fermentation nutrient solution, wherein the microbial fermentation nutrient solution is obtained by fractionally fermenting a fermentation substrate with a compound microbial agent and then filtering to obtain the clear liquid at the top layer; The compound microbial agent is composed of the following strains in parts by weight: 20-30 parts of Bacillus subtilis, 25-35 parts of Lactobacillus plantarum, 15-25 parts of yeast, and 10-20 parts of Bacillus megaterium; the total effective viable count of the compound microbial agent is ≥2.0 × 10⁻⁶. 9 CFU / g; The fermentation substrate consists of the following components by mass percentage: 1.5%–3% glucose, 2%–4% soybean meal hydrolysate, 0.08%–0.15% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, and the balance being water.

[0008] In a further preferred embodiment, the composite microbial agent performs segmented fermentation of the fermentation substrate as follows: first, aeration fermentation at 28–30°C for 24 hours, followed by micro-anaerobic static fermentation at 25–27°C for 48–72 hours; the β-nicotinamide mononucleotide content in the microbial fermentation nutrient solution is ≥120 mg / L, and the free oxidized nicotinamide adenine dinucleotide content in the microbial fermentation nutrient solution is <5 mg / L.

[0009] More preferably, the microbial fermentation nutrient solution has a pH value of 5.8 to 6.5 and a total amino acid content of ≥1.2 g / L.

[0010] Further preferably, the Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus megaterium are all wild-type strains that have not undergone genetic engineering modification.

[0011] The method for preparing the above-mentioned microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide includes the following steps: S1. Activation of microbial strains: Add the compound microbial agent to the sterile activation solution and culture with shaking at 28-32℃ for 12-16 hours to obtain the seed culture; S2. Fermentation substrate preparation: according to the ratio, dissolve glucose, soybean hydrolysate, nicotinamide, magnesium sulfate and potassium dihydrogen phosphate in water, adjust the pH to 6.0-6.8, sterilize at high temperature and then cool to room temperature to obtain the fermentation substrate; S3. Subsection fermentation: according to the volume ratio of fermentation substrate to seed liquid 1:0.08-0.12, the seed liquid obtained in step S1 is added to the fermentation substrate obtained in step S2, first aerated fermentation at 28-30℃ for 24h, then micro-anaerobic static fermentation at 25-27℃ for 48-72h to obtain the fermentation liquid; S4. Separation and liquid collection: filter the fermentation liquid obtained in step S3 to remove bacterial residue, and collect the upper clear liquid after standing and settling to obtain the microbial fermentation nutrient liquid rich in β-nicotinamide mononucleotide.

[0012] Further preferably, the high-temperature sterilization in step S2 is performed at 115℃ for 20 min.

[0013] Further preferably, in step S4, an 80-mesh filter is used for filtration, and the standing and settling time is 10-14h.

[0014] Further preferably, in step S3, the aeration ratio of aeration fermentation is 1:0.5-1:1.5 (v / v·min), and the dissolved oxygen concentration is controlled to be <1 mg / L during micro-anaerobic static fermentation.

[0015] The application method of the microbial fermentation nutrient liquid rich in β-nicotinamide mononucleotide is to use the microbial fermentation nutrient liquid for foliar spraying or root irrigation or drip irrigation of crops; when foliar spraying, the microbial fermentation nutrient liquid is diluted 500-800 times before use, and is sprayed at the seedling stage, fruit expansion stage or high-temperature stress stage of crops, and is sprayed once every 7-10 days, and is used continuously for 2-3 times; when root irrigation or drip irrigation, the microbial fermentation nutrient liquid is diluted 300-500 times before use, and is flushed with water.

[0016] By adopting the foregoing technical solutions, the present application has the following beneficial effects: the present application successfully prepares a microbial fermentation nutrient liquid rich in high-content β-nicotinamide mononucleotide (NMN) by using a conventional safe strain which is not genetically engineered, a fermentation substrate formula and subsection fermentation, that is, by selecting multiple strains of metabolically complementary complex functional bacteria, regulating the carbon-nitrogen ratio, temperature, dissolved oxygen and fermentation time, blocking the excessive conversion of NMN to downstream products, and allowing a large amount of NMN to be secreted to the upper clear liquid of the fermentation; the small molecule β-nicotinamide mononucleotide (NMN) in the fermentation liquid is absorbed by the roots and leaves of crops, and is converted to endogenous NAD in plant cells. +Coenzyme, up-regulate antioxidant enzyme activity, improve energy metabolism efficiency, relieve stress damage, delay plant senescence. Fermentation by-products organic acids, free amino acids, microbial polysaccharides, active peptides synergistic effect, improve rhizosphere microecology, promote root growth and rooting. After application, it can significantly improve the endogenous NAD + content in plant cells, enhance stress resistance, delay early senescence, and improve crop yield and quality. Specifically: 1. Four conventional agricultural microorganisms, Bacillus subtilis, Lactobacillus plantarum, yeast and Bacillus megaterium, are wild type strains without genetic engineering, with high biological safety, meeting the requirements of organic agricultural inputs, and can be mass produced and popularized.

[0017] 2. The fermentation substrate raw materials are by-products of bulk agricultural product processing (soybean meal hydrolysate) and conventional chemicals (glucose, nicotinamide, etc.), which are widely available and low in price. The fermentation process is simple and does not require complex equipment, with production cost much lower than that of chemical synthesis and enzyme catalysis.

[0018] 3. Through the metabolic complementation and synergistic effect of four bacteria and the time sequence regulation of the segmented fermentation process, the NMN content in the upper layer of the clarified liquid is ≥120 mg / L, which is significantly higher than that of single strain fermentation and existing natural strain fermentation.

[0019] 4. NMN has small molecular weight and good water solubility, which can be directly and efficiently absorbed by crop roots and leaves, overcoming the defects of exogenous NAD + with large molecular weight, difficult to penetrate cell membrane and low absorption efficiency. After crop absorption of NMN, endogenous NAD + is rapidly converted in plant cells through NAD + supplemental synthesis pathway, effectively improving cell energy metabolism and antioxidant capacity.

[0020] 5. The free NAD + content in the upper layer of the clarified liquid is very low (<5 mg / L), NMN exists in a stable form, has low storage loss (decrease <8% in 90 days) at room temperature and in the dark, and is convenient for field application, suitable for foliar spraying, root irrigation, drip irrigation and other application scenarios. It can simultaneously improve crop stress resistance (drought resistance, high temperature resistance, disease resistance), delay leaf senescence, and improve fruit yield and quality. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application.

[0022] The terms "first", "second", "third" and the like in the description and claims of the present application are used for distinguishing between similar objects having a same name and not necessarily for describing a specific sequential order. Moreover, the terms "comprising", "having", "including" and "containing" or any other similar forms are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises, has, includes or contains an item or list of items does not require that any of the items or lists of items be present or be necessarily the only items or lists of items. As used herein, the terms "about" and "substantially" are used to allow for any variations that can exist in, for example, measured, calculated, estimated or experimental quantities in the art that are normal, allowable or acceptable. Unless otherwise specified, all raw materials referred in the examples are commercially available, and all operation steps are conventional operations in the art; all detection methods are corresponding industry standards or conventional detection methods, wherein the effective viable cell count is detected by plate spreading method, the total nutrient is detected by weight method, the miscellaneous bacteria rate is detected by plate counting method, the functional ingredient is detected by high performance liquid chromatography (HPLC), the osmotic pressure is detected by osmotic pressure instrument, and the pH value is detected by pH meter.

[0023] The Bacillus subtilis used in the present application is a strain with the preservation number CGMCC No. 1.1091; the Lactobacillus plantarum is a strain with the preservation number CGMCC No. 1.1856; the Saccharomyces cerevisiae is a strain with the preservation number CGMCC No. 2.396; and the Bacillus megaterium is a strain with the preservation number CGMCC No. 1.7397. The above strains can be purchased through commercial channels such as China General Microbiological Culture Collection Center (CGMCC).

[0024] The functions of each strain in the complex microbial inoculant in the present application are as follows: (1) Bacillus subtilis The Bacillus subtilis has nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) activity, which is responsible for converting nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) into β-nicotinamide mononucleotide (NMN). A certain level of NMN (about 53.67 mg / L) can be detected in the fermentation system containing nicotinamide, and the endogenous nicotinic acid phosphoribosyltransferase (PncB) of the Bacillus subtilis is a key enzyme for NMN synthesis. In addition, the protease secreted by the Bacillus subtilis can decompose macromolecular proteins in the soybean meal hydrolysate into small molecular peptides and free amino acids, thereby providing nitrogen source for other strains.

[0025] (2) Lactobacillus plantarum *Lactobacillus plantarum* is a lactic acid bacterium with the ability to convert nicotinamide to NMN via the nicotinamide nucleoside kinase (NRK) pathway. The lactic acid produced by its metabolism can lower the pH of the fermentation environment, creating an acidic microenvironment (pH 5.8–6.5) conducive to NMN accumulation. Simultaneously, this acidic environment can inhibit the activity of NMN-degrading enzymes (such as CD38 and CD157 exonucleases), reducing the conversion of NMN to nicotinamide adenine dinucleotide (NAD). + Excessive conversion of ).

[0026] (3) Yeast (Saccharomyces cerevisiae) Yeast has a high efficiency in NAD. + The salvage synthesis pathway can further convert NMN into NAD. + However, in the second stage of staged fermentation (microanaerobic settling, 25–27°C, DO < 1 mg / L), by controlling dissolved oxygen and temperature conditions, the yeast shifted from "respiratory metabolism" to "fermentation metabolism," and its NMN→NAD conversion... + The conversion activity was significantly inhibited, allowing NMN to accumulate in large quantities in the fermentation broth instead of being further consumed.

[0027] (4) Bacillus megaterium Bacillus megaterium possesses phosphorus and potassium solubilizing capabilities, converting organic phosphorus in the fermentation substrate into inorganic phosphorus and providing phosphate donors (PRPP) for NMN synthesis. Simultaneously, the organic acids and extracellular polysaccharides produced by its metabolism help improve the rhizosphere microecological environment of plants.

[0028] The synergistic mechanism of the four-strain combination: Bacillus subtilis provides the NAMPT pathway for direct NMN synthesis; Lactobacillus plantarum provides the NRK pathway and regulates the pH environment to inhibit NMN degradation; yeast acts as an NMN / NAD... + The four strains act as a regulatory hub for metabolism (controlling metabolic direction through a segmented fermentation process); Bacillus megaterium provides phosphate donors and improves the inorganic nutrition of the fermentation system. They complement each other in substrate utilization, metabolite production, and fermentation environment regulation, achieving highly efficient NMN accumulation. Example 1

[0029] 1. Compound microbial agent formulation (parts by weight) 25 samples of Bacillus subtilis, 30 samples of Lactobacillus plantarum, 20 samples of yeast, and 15 samples of Bacillus megaterium; total effective viable count 2.5 × 10⁻⁶. 9 CFU / g.

[0030] 2. Fermentation substrate formulation (by weight percentage) The ingredients are: 2% glucose, 3% soybean meal hydrolysate, 0.12% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, and the remainder is water.

[0031] 3. Preparation method S1. Strain activation: Add the compound microbial agent to the sterile activation solution and culture it with shaking at 30°C for 14 hours to obtain the seed culture; S2. Preparation of fermentation substrate: Dissolve each raw material in water according to the ratio, adjust the pH to 6.5, sterilize at 115℃ for 20 min, cool to room temperature, and obtain the fermentation substrate; S3. Segmented fermentation: The seed liquid was inoculated into the fermentation substrate at a volume ratio of 1:0.1 between the fermentation substrate and the seed liquid. The fermentation was first carried out at 29℃ with aeration for 24 h (aeration ratio 1:1 v / v·min), and then carried out under micro-anaerobic static fermentation at 26℃ for 60 h (DO < 1 mg / L) to obtain the fermentation broth. Under aeration conditions (ventilation ratio 1:0.5–1:1.5 v / v·min), aerobic bacteria (Bacillus subtilis, Bacillus megaterium) and facultative bacteria (yeast) proliferate rapidly, expressing large amounts of NMN synthesis-related enzymes (NAMPT, NRK, etc.). Simultaneously, they consume glucose in the fermentation substrate to produce ATP and PRPP, providing energy and phosphoribose donors for NMN synthesis. This stage is dominated by cell proliferation and enzyme expression. After aeration was stopped and the temperature was lowered, the fermentation system entered a micro-anaerobic state (dissolved oxygen DO < 1 mg / L). During this stage, aerobic bacteria proliferated slowly, and metabolism shifted to product accumulation; *Lactobacillus plantarum* became the dominant bacteria, continuously producing lactic acid to lower the pH; under micro-anaerobic conditions, yeast shifted from "respiratory metabolism" to "fermentation metabolism," converting NMN to NAD. + The conversion activity is inhibited, allowing NMN to accumulate in large quantities in the fermentation broth instead of being further consumed; S4. Separation and liquid collection: Filter the fermentation broth through an 80-mesh filter to remove bacterial residues, let it stand for 12 hours to settle, and collect the clear liquid at the top to obtain the microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide.

[0032] HPLC method for determining β-nicotinamide mononucleotide (NMN) content: Chromatographic column: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); Mobile phase: 20mM potassium dihydrogen phosphate buffer (pH 6.0):methanol = 95:5 (v / v); Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 260nm; Injection volume: 20 μL; Retention time: Approximately 6.5 minutes for NMN standard.

[0033] 4. Product performance testing The finished product pH was 6.2; the β-nicotinamide mononucleotide content in the clarified upper layer of fermentation liquid was 186 mg / L; free NAD+... + The content was 3.2 mg / L; the total amino acid content was 1.8 g / L.

[0034] 5. Field trials Test crop: Tomato (variety: Provence). During the fruit expansion period, the microbial fermentation nutrient solution prepared in Example 1 was diluted 600 times and sprayed on the leaves. It was sprayed once every 7 days for 3 consecutive times. The same amount of water was sprayed as a blank control.

[0035] Detection results: Endogenous NAD in tomato leaf cells of the treatment group + The chlorophyll content was 68% higher than that of the water control group; the leaf senescence of the treatment group was delayed, and the chlorophyll content was 23% higher than that of the control group; the single fruit weight of the treatment group was 16.3% higher than that of the control group, and the soluble solids content was 2.1 percentage points higher. Example 2

[0036] 1. Compound microbial agent formulation 20 samples of Bacillus subtilis, 35 samples of Lactobacillus plantarum, 15 samples of yeast, and 20 samples of Bacillus megaterium; total effective viable count 2.2 × 10⁻⁶. 9 CFU / g.

[0037] 2. Fermentation substrate formulation (by weight percentage) The ingredients are: 1.5% glucose, 4% soybean meal hydrolysate, 0.08% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, and the remainder is water.

[0038] 3. Preparation method S1. Activation of microbial strains: Add the compound microbial agent to the sterile activation solution and incubate with shaking at 28°C for 16 hours to obtain the seed culture; S2. Fermentation substrate preparation: Dissolve each raw material in water according to the ratio, adjust the pH to 6.0, sterilize at 115℃ for 20 minutes, cool to room temperature, and obtain the fermentation substrate; S3. Segmented fermentation: The seed liquid was inoculated into the fermentation substrate at a volume ratio of 1:0.08 between the fermentation substrate and the seed liquid. The fermentation was first carried out at 28℃ with aeration for 24 h (aeration ratio 1:0.8 v / v·min), and then carried out under micro-anaerobic static fermentation at 25℃ for 72 h (DO < 1 mg / L) to obtain the fermentation broth. S4. Separation and liquid collection: Filter the fermentation broth through an 80-mesh filter to remove bacterial residues, let it stand for 14 hours to settle, and collect the clear liquid at the top to obtain the microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide.

[0039] 4. Product performance testing The finished product pH was 6.0; the β-nicotinamide mononucleotide content in the clarified upper layer of fermentation liquid was 152 mg / L; free NAD+... + The content was 2.8 mg / L; the total amino acid content was 1.5 g / L.

[0040] 5. Field trials Test crop: Tomato (variety: Provence). During the fruit expansion period, the microbial fermentation nutrient solution prepared in Example 2 was diluted 500 times and sprayed on the leaves. It was sprayed once every 7 days for 3 consecutive times. The same amount of water was sprayed as a blank control.

[0041] Detection results: Endogenous NAD in tomato leaf cells of the treatment group + The chlorophyll content was 71% higher than that of the water control group; the leaf senescence of the treatment group was delayed, and the chlorophyll content was 25% higher than that of the control group; the single fruit weight of the treatment group was 17.3% higher than that of the control group, and the soluble solids content was 2.2 percentage points higher. Example 3

[0042] 1. Compound microbial agent formulation 30 samples of Bacillus subtilis, 25 samples of Lactobacillus plantarum, 25 samples of yeast, and 10 samples of Bacillus megaterium; total effective viable count 2.8 × 10⁻⁶. 9 CFU / g.

[0043] 2. Fermentation substrate formulation (by weight percentage) The ingredients are: 3% glucose, 2% soybean meal hydrolysate, 0.15% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, and the remainder is water.

[0044] 3. Preparation method S1. Activation of microbial strains: Add the compound microbial agent to the sterile activation solution and incubate with shaking at 32°C for 12 hours to obtain the seed culture; S2. Fermentation substrate preparation: Dissolve each raw material in water according to the ratio, adjust the pH to 6.8, sterilize at 115℃ for 20 minutes, and cool to room temperature to obtain the fermentation substrate; S3. Segmented fermentation: The seed liquid was inoculated into the fermentation substrate at a volume ratio of 1:0.12 between the fermentation substrate and the seed liquid. The fermentation was first carried out at 30℃ with aeration for 24 h (aeration ratio 1:1.5 v / v·min), and then carried out under micro-anaerobic static fermentation at 27℃ for 48 h (DO < 1 mg / L) to obtain the fermentation broth. S4. Separation and liquid collection: Filter the fermentation broth through an 80-mesh filter to remove bacterial residues, let it stand for 10 hours to settle, and collect the clear liquid at the top to obtain a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide.

[0045] 4. Product performance testing The finished product pH value was 6.4; the β-nicotinamide mononucleotide content in the clarified liquid at the top of fermentation was 135 mg / L; free NAD+... + The content was 4.1 mg / L; the total amino acid content was 1.3 g / L.

[0046] 5. Field trials Test crop: cucumber (variety: Jinyou No. 1). Select plants with uniform growth and in the seedling stage. Spray the leaves with the microbial fermentation nutrient solution prepared in Example 3 diluted 600 times. Spray once every 7 days for 2 consecutive times. Spray with an equal amount of water as a blank control.

[0047] Detection results: Endogenous NAD2 was collected from functional leaf cells of the same part of plants in each treatment group. + The content was 63% higher than that of the water control group; the yield per plant increased by 12%; and the vitamin C content increased by 10%.

[0048] Comparative Example 1 (fermentation with a single Lactobacillus plantarum) Fermentation was carried out using only Lactobacillus plantarum (the same number of viable bacteria as in Example 1), with the same fermentation substrate and preparation method as in Example 1.

[0049] Test results: The NMN content in the clarified liquid at the top of the fermentation layer was only 28 mg / L. Field trials (under the same conditions as Example 1) showed that the endogenous NAD content in the tomato leaves of the treatment group was... + The increase was only 12%, and the increase in single fruit weight was not significant.

[0050] Comparative Example 2 (fermentation with a single Bacillus subtilis) Fermentation was carried out using only Bacillus subtilis (the same number of viable bacteria as in Example 1), with the same fermentation substrate and preparation method as in Example 1.

[0051] Test results: The NMN content in the clarified liquid at the top of the fermentation layer was only 35 mg / L. Field trials (under the same conditions as Example 1) showed that the endogenous NAD content in the tomato leaves of the treatment group was... + An increase of only 15% is not a significant increase in production.

[0052] Comparative Example 3 (without using a segmented fermentation process) The same compound microbial agent formula and fermentation substrate formula as in Example 1 were used, except that segmented fermentation was not used, but continuous aeration fermentation was carried out at a constant temperature of 29°C for 84 hours (the total fermentation time was the same as 24h+60h=84h in Example 1).

[0053] The results showed that the NMN content in the clarified liquid at the top of the fermentation layer was 62 mg / L, significantly lower than the 186 mg / L in Example 1. This indicates that the staged fermentation process plays a crucial role in the targeted enrichment of NMN—initial aeration promotes cell proliferation and enzyme expression, while subsequent micro-anaerobic settling facilitates NMN accumulation and inhibits its conversion to NAD+. + Excessive conversion.

[0054] Comparative Example 4 (no nicotinamide precursor added to the fermentation substrate) The same compound microbial agent formulation and preparation method as in Example 1 were used, except that nicotinamide was not added to the fermentation substrate.

[0055] The test results showed that the NMN content in the clarified liquid at the top of the fermentation layer was 18 mg / L. This indicates that the exogenous addition of nicotinamide precursor is a necessary condition for high NMN production, and that microorganisms convert nicotinamide into NMN through a salvage synthesis pathway.

[0056] Product stability test The microbial fermentation nutrient solution prepared in Example 1 was sealed and stored at room temperature and away from light for 90 days. The NMN content was retested and found to be 171 mg / L, a decrease of 8.1%; free NAD... + The concentration was 3.8 mg / L, with no significant increase. This indicates that NMN exists in a stable form in the product and has not been largely converted into NAD. + It has good storage stability.

[0057] Plant endogenous NAD + Content detection test The test crop was cucumber (variety: Jinyou No. 1). Three treatment groups were set up: (1) a blank control group with water; (2) a control group with commercially available ordinary microbial fertilizer (diluted 500 times); and (3) the experimental group of microbial fermentation nutrient solution of Example 1 of this invention (diluted 600 times). Foliar spraying was carried out uniformly during the seedling stage. Functional leaves were collected 72 h later, and the intracellular NAD⁺ concentration was detected by high performance liquid chromatography-mass spectrometry (LC-MS).

[0058] Detection results: Endogenous NAD in cucumber leaf cells of the experimental group + The content was 72% higher than the water-based blank control group and 58% higher than the control group of commercially available ordinary microbial fertilizer. This proves that the NMN in the microbial fermentation nutrient solution of this invention can be effectively absorbed by crop leaves and converted into endogenous NAD. + .

[0059] Application method instructions The microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide prepared in the above embodiments can be used for foliar spraying, root irrigation, or drip irrigation of crops. For foliar spraying, the microbial fermentation nutrient solution is diluted 500-800 times before use, and sprayed during the seedling stage, fruit expansion stage, or high-temperature stress period, preferably once every 7-10 days, for 2-3 consecutive applications. For root irrigation or drip irrigation, the microbial fermentation nutrient solution is diluted 300-500 times before use, and applied with irrigation water. The microbial fermentation nutrient solution is suitable for fruit and vegetable crops such as tomatoes, cucumbers, peppers, eggplants, and strawberries; grain crops such as rice, wheat, and corn; and cash crops such as tea, cotton, and medicinal herbs.

[0060] It should be noted that Examples 1-3 above used three different ratios (25:30:20:15, 20:35:15:20, 30:25:25:10) of Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus megaterium within the scope of claim 1, as well as different combinations of the contents of the three components in the fermentation substrate. All of these combinations successfully prepared microbial fermentation nutrient solutions with an NMN content ≥120 mg / L, demonstrating that the technical solution of this invention has good consistency and reproducibility within the scope of claim 1. Those skilled in the art, within the scope defined by claim 1, can adjust the amounts of each component according to actual needs without creative effort and expect to obtain the same technical effects.

[0061] The microbial fermentation nutrient solution prepared by this invention should be sealed and stored at room temperature (≤30℃) in the dark. High temperatures (>40℃) and direct sunlight should be avoided to prevent photothermal degradation of NMN. The viable cell ratio of Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus megaterium is (2-3):(2.5-3.5):(1.5-2.5):(1-2)×10⁻¹⁰. 8 The CFU / g ratio is preferred. The compound microbial agent preferably consists of the following strains in parts by weight: 20-30 parts Bacillus subtilis, 25-35 parts Lactobacillus plantarum, 15-25 parts yeast, and 10-20 parts Bacillus megaterium; the total effective viable count of the compound microbial agent should be ≥2.0 × 10⁻⁶. 9 The optimal CFU / g ratio is preferred. The fermentation substrate should preferably consist of the following components by mass percentage: 1.5%–3% glucose, 2%–4% soybean meal hydrolysate, 0.08%–0.15% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, with the balance being water.

[0062] Although the invention has been specifically shown and described in conjunction with preferred embodiments, those skilled in the art should understand that various changes in form and detail may be made to the invention without departing from the spirit and scope of the invention as defined in the appended claims, all of which shall be within the scope of protection of the invention.

Claims

1. A microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide, comprising a microbial fermentation nutrient solution, characterized in that: The microbial fermentation nutrient solution is prepared by fermenting the fermentation substrate in stages with a compound microbial agent, followed by filtration to obtain the clear liquid from the upper layer. The compound microbial agent is composed of the following strains in parts by weight: 20-30 parts of Bacillus subtilis, 25-35 parts of Lactobacillus plantarum, 15-25 parts of yeast, and 10-20 parts of Bacillus megaterium; the total effective viable count of the compound microbial agent is ≥2.0 × 10⁻⁶. 9 CFU / g; The fermentation substrate consists of the following components by mass percentage: 1.5%–3% glucose, 2%–4% soybean meal hydrolysate, 0.08%–0.15% nicotinamide, 0.02% magnesium sulfate, 0.03% potassium dihydrogen phosphate, and the balance being water.

2. The microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 1, characterized in that: The compound microbial agent is used to ferment the fermentation substrate in stages: first, it is fermented with aeration at 28-30℃ for 24 hours, and then it is fermented under micro-anaerobic conditions at 25-27℃ for 48-72 hours. The content of β-nicotinamide mononucleotide in the microbial fermentation nutrient solution is ≥120 mg / L, and the content of free oxidized nicotinamide adenine dinucleotide in the microbial fermentation nutrient solution is <5 mg / L.

3. The microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 1, characterized in that: The microbial fermentation nutrient solution has a pH value of 5.8–6.5 and a total amino acid content of ≥1.2 g / L.

4. The microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 1, characterized in that: The Bacillus subtilis, Lactobacillus plantarum, yeast, and Bacillus megaterium mentioned are all wild-type strains that have not undergone genetic engineering modification.

5. A method for preparing a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to any one of claims 1 to 4, characterized in that: Includes the following steps: S1. Activation of microbial strains: Add the compound microbial agent to the sterile activation solution and culture with shaking at 28-32℃ for 12-16 hours to obtain the seed culture; S2. Preparation of fermentation substrate: Dissolve glucose, soybean meal hydrolysate, nicotinamide, magnesium sulfate and potassium dihydrogen phosphate in water according to the specified ratio, adjust the pH to 6.0-6.8, sterilize at high temperature and cool to room temperature to obtain the fermentation substrate; S3. Segmented fermentation: The seed liquid obtained in step S1 is inoculated into the fermentation substrate obtained in step S2 at a volume ratio of 1:0.08 to 0.12 between the fermentation substrate and the seed liquid. The fermentation is first carried out at 28 to 30°C for 24 hours with aeration, and then carried out at 25 to 27°C for 48 to 72 hours under micro-anaerobic static fermentation to obtain the fermentation liquid. S4. Separation and liquid collection: Filter the fermentation broth obtained in step S3 to remove bacterial residues, let it stand to settle, and collect the clear liquid at the top to obtain a microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide.

6. The method for preparing the microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 5, characterized in that: In step S2, high-temperature sterilization is performed at 115°C for 20 minutes.

7. The method for preparing microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 5, characterized in that: In step S4, an 80-mesh filter is used for filtration, and the settling time is 10-14 hours.

8. The method for preparing the microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to claim 5, characterized in that: In step S3, the ventilation ratio for aeration fermentation is 1:0.5 to 1:1.5 (v / v·min), and the dissolved oxygen concentration is controlled to be <1 mg / L during micro-anaerobic static fermentation.

9. A method for applying the microbial fermentation nutrient solution rich in β-nicotinamide mononucleotide according to any one of claims 1 to 4, characterized in that: The microbial fermented nutrient solution can be used for foliar spraying, root irrigation, or drip irrigation of crops. For foliar spraying, dilute the microbial fermented nutrient solution 500-800 times before use, and spray during the seedling stage, fruit expansion stage, or high temperature stress period of crops, once every 7-10 days, for 2-3 consecutive applications. For root irrigation or drip irrigation, dilute the microbial fermented nutrient solution 300-500 times before use, and apply it with water.